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FlowJo Advanced Tutorial - Flow Cytometry Core Facility

1. Ka FlowJo Layouts Advanced 1utonial Chapter 10 wsp Eile Edit Layout Object View Align Help NJ OS BBY AY A AJ 100 o a3 off Resize the table so that most of the text is shown in each cell left Statistics Samples Freq of P Freq ofPi Freq of Pj Freq ofPi Freq of P BI 78 06 60 67 30 51 47 53 20 10 If you increase the magnification on the Layout Editor you should see your table similar to the one show here Statistics Samples Freq o P Freq ofPi Freq ofP Freq of Pi 931115 B 78 06 60 67 30 51 47 53 931115 c1 78 99 73 06 29 30 65 55 931115 DI 61 08 67 75 50 97 45 63 931115 E 63 33 62 01 68 02 23 12 82 Now it s time to create a Grid In this Grid you will have three rows one title row one for CD4 T cells and one for CD8 T cells As well you will have 4 columns the first is a title the second will have the Median CD3 fluorescence on the specified subset the third will have the percent of CD3 T cells that are CD4 or CD8 and the fourth will have a histogram of the CD3 intensity for the gated subset I FlowJo Layouts Advanced Tutorial Chapter 10 wsp File Edit Layout Object View Align Help 5 Se a a ajr To create a blank Grid click on the Grid tool icon it is the fourth from left at the top left edge of the layout window Click xla and drag a rectangle that occupies the right portion of the Gray Statistics pa
2. ED Lymphocytes you have more than a few changes TDCS Naw ai Ch attshic Falls Instead copy the whole tree wo 931115 B08 Sample 01 fes 10000 55 Lymphocytes T044 rada Go COAT 24 rO 2470 Ed 6 42 T CySPE COS Median 75 26 E Freq of Parent 25 99 v Me 362 CySPE COB Median 64 83 2 Freq of Pareni 14 56 wr E WG 649 E CySPE COS Median E Freq of Parent wD Nawe E CySPE CDE Median Freq of Parani 991115 C08 Sample 02 fes w OED Lymphocytes wif COAT ED MI ED M2 ED Ma E Naive 931115 D08 Sample 03 fes w OED Lymphocytes wf COaT E Mi Get M2 ED Ma ED Nawa U 931115 E08 Sample 04 ce w ED Lymphocytes D caT ED M ED M2 File Edit Workspace Groups Tools Windows Help Debug Dj Ee S Click on the Lymphocyte subset under roups a nalyses umber of Samples agg PP es is ioe 1 the sample named 93115 B08 and eos su while holding the Alt key to drag the Ye coer entire tree drop it onto the C08 sample v TM e E CYPE Median Make sure you drop it onto the sample _ 2 Freq of Parent s aT level that is where it needs to be Tati 808 Semple O18 SS attached You can also copy the tree to SO ome oo the CD8 Group to add the new statistics ci oars to all samples in the group FlowJo TON crm L 323 assumes that when you drag an analysis Ca ans ese tree to a group you want to replace all ieee Tr of the existing analyses that have the
3. Population Name Monocytes O E m 2 Z i FhyEre CD16 6l Click on the button and name the new Layout Contour Analysis Now select the Lymphocyte subset from the first sample and drag it into the Layout Editor Fy FlowJo Layouts Tutorial WS Chapter 6 wsp File Edt Layout Object View Align Help Kjojsiejajaj ajr vje SE rjsl i Contour Analysis Contour Analysis Copy Paste Duplicate Properties Ordering Remove Data Hide Annotation Show Legend Show Agjunct Histograms Show Ancestry with Backgating Make MultiGraph Overlay 50 Farge G31115 607 Sample 01 fes Lymphocytes Tags Because this plot is a subset two additional options appear in the menu Show Ancestry and With Backgating Right click the layout to see these options With the first you can display subset plots showing the population s ancestry FlowJo shows you not only the graph for a subset but the plots of all parent populations as well including their backgating if you so desire GON BOTs ampk OT fr Lyrmphesyees COPE COM FlowJo gives you enormous flexibility in designing customized layouts so that you can quickly and easily generate high quality publication and presentation graphics 62 Lesson 8 Creating Batch Graphical Reports In this chapter you will build on what you learned in Chapter 7 to generate graphical reports for entire experiments You will learn how to cycle the Layout
4. Fight in Top ins Bethe in With File gt Print in the Layout Editor you can change the Page Setup to orient the page and change the paper size and page margins to your needs The dashed gray lines drawn in the Layout View represent the edge of the printable area to view these lines 55 click View from Layout menu bar and choose Show Page Breaks you can drag these at any intersection to adjust the print area per page of your layout Page margins are added outside these lines when printing You can also copy items from layout to layout within the same workspace Click on the first graphic only and choose Copy from the Edit menu or press Ctrl C You can now create a new layout and paste that object into the new layout As noted before you can have as many different layouts in each Workspace as you wish To create a new layout click on the button near the top left of the window To delete a layout click on the button File Edit Layout Object KJjojnjejaje Scatter Analys Untitled Now let s create a second layout to demonstrate how to create graph overlays Create a new Layout and name it Overlay Drag the first Lymphocytes subset from 9 3 1115 B01 and drop it in the right hand pane of the Layout window You should have a view of the CD45 histogram as shown here IE FlowJo Layouts Tutorial WS Chapter 7 wsp File Edit Layout Object View Align Help gaat Bajo h Dae i S
5. Underneath the sample you just gated indented one level is a new row This new row represents the subpopulation defined by the gate This row begins with a subset icon followed by the name that you gave to the subset To the right you will find the frequency of these events within the sample and the total number of events in this gate Any operation that can be performed on a sample can also be done to a subpopulation You can double click on the 0 Lymphocytes subset to open its graph gate within that subset etc Double click on this population You will see the graph at right This is the contour plot for the events falling within the lymphocyte gate Note that the number of events in this graph Count at the bottom of the window is 7100 This is the number of cells falling within the lymphocyte gate You will also see that the Up arrow button is now active DER ra Clicking on this button opens the graph you Kili used to create this subset i e you will see the gate for this subset The Up arrow can be used to navigate to the parent population the Down arrow navigates to the subset Click on the down arrow or double click on the gate in the graph to show a Lymphocytes 931115 B01 Sample 01 fc File Edit Graph Display Go Help gt Options new Graph window for the subset b Active Gate gt Statistics Count 7100 10000 71 0 19 Change this graph to CD14 vs CD45 your graph i a ee
6. FlowJo copies the analysis into the new location Now it is time to analyze another set of cells from this same patient This lesson builds on the Workspace you finished in Chapter 2 alternatively you can open the workspace named Tutorial WS Chapter 3 wsp Browse to the folder Experiment 1 and select the first sample Click OK and the workspace will be populated with 16 samples four sets of PBMC with four stains each We will analyze the second file in the Workspace which has the sample from patient one stained with the second combination of reagents Here is where you will get the first glimpse of the powerful batch analyses that FlowJo can perform You have already made a lymphocyte gate on stain 1 presumably this same gate should be applied to stain 2 To do this simply click on the Lymphocyte subset in the Workspace window and drag it down so that the second sample 9931115 B02 Sample 01 fcs is highlighted then let go of the mouse FlowJo creates a new subpopulation using the same gate you had previously created 1 20090303 1 FlowJo Your Workspace window NOW _ Elle Edt Workspace Groups Tools Windows Help Debug looks like the one on the right Ti z a LS Groups and Analyses Number of Samples 16 T All Samples u Experiment 1 16 E Lymphocytes 931115 B02 Sample 01 fc LAX Nae e Wu 5 B01 Sample 01 fcs w d Lymphocytes 71 00 7100 w ED CD45 63 80 6380 Y Cy5PE CD45 CV 35 27 Y Cy5PE CD45 M
7. FlowJo again asks you the name for this new subpopulation subset of cells Type in CD45 as the name of the subset Histogram The Graph window now displays the gate and the statistics for that gate as shown left The vertical placement of the gate is irrelevant you can drag the gate L KA horizontally or vertically by clicking on the e e ee horizontal bar the cursor becomes a hand and moving it around You can change the upper or lovver boundaries by clicking on one of the handles and moving it The Workspace window now has a new entry as shown in the example on the next page k Options gt Active Gate CD45 CD45 63 50 p Statistics Count 7100 10000 71 0 21 E 20090303 1 FlowJo File Edit Workspace Groups Tools Windows Help Debug T 2 Groups and Analyses Note that the new entry is below Lymphocytes and is further indented This is because the new population is a subset of Lymphocytes The Workspace reflects this hierarchy The numbers to Number of Samples i All Samples 16 1 Experiment 1 16 Name dh Statistic T Cells cySPE reagent Fluor reagent Phyery reagent the E ight indicate the 931115 B01 Sample 01 fes 10000 aj fraction of events falling m eheys fee an within the gates Thus 7 f 931115 B02 Sample 01 fes 10000 CD4 cD3 CD8 71 0 of the sample s u 931115 BO7 Sample 01 fes 10000 Cp4 L se
8. and click on the Table button ai Lymphocytes Freq of Parent req of Pare i a Lymphocytes CD8TIM3 Freq ofParent 1 e orat 1 Remember you can apply a table gt GproctesDSTIMI Freg Paet mdha template to any group this could be i ass armi useful for separately analyzing different experiments that you have grouped separately If you apply a table to a group that has no gates as E Lymphoctesfe08 TN pe I Gj requested by the table you will get a J E O a t lot of empty values Ie E Freg of Parent 11 Lymphocytes CD8 T Naive Median CSPE Median CySFE d2 Lymphocytes CD8 TIME Median CSPE Median CysPEJ 48 Tables can also be added to Layouts in the Layout Editor Simply use the generated Table menu item FlowJo gt Add to Layout in a generated table window and FlowJo will create a live table that updates as you create batch graphical layouts Table definitions are saved with the Workspace When you re open the Workspace you can recalculate the tables Tables can be applied to any group you can in the future read more samples into the Workspace and apply the table to those samples Table templates are a useful batch analysis tool they allow you to quickly collate many statistics from many different samples for analysis by other programs 49 Lesson 7 Creating Simple Graphical Layouts In this chapter you will learn the fundamentals of the Layout Editor
9. s se osiy co nep is automatically updated To see this in action switch your HHY Job attention back to the workspace and double click on the 931115 B01 sample node You should see the graph to the N _ right defining the gates for the chilean Lymphocytes and Monocytes Kjoj sjejz opa subsets Click on the Monocytes se z gate upper gate and move it around perhaps as shovyn on the left to overlap with the Lymphocytes subset ForSc gt Options b Active Gate Monocytes Monocytes22 0096 gt Statistics Count 10000 10000 100 096 Forsc Monocytes Monocytes34 51 Count 10000 10000 100 0 58 Note that the Layout Editor responds by updating the green dots corresponding to Monocytes see below The order in which the subsets are drawn can have a significant effect on what the graphs look like To change the order click on any subset name in the legend and drag it up or down the cursor changes shape to let you know what is happening For example click on the Lymphocytes subset and move it to the top Note that the area which has both green and red dots now appears to be completely red because the red population is on top i e drawn last see the example below FlowJo Layouts Tutorial WS Chapter 7 wsp File Edit Layout Object View Align Help WEINE aja aj 100 vi aj E Overlay Scatter Analysis i D fu u CyYSPE CD45 59 FlowJo L
10. s selection only applies to workspaces you create subsequently it doesn t affect the gt Active Gate currently open graphs Almost every window gt Statistics Count 10000 10000 100 0 in FlowJo has the Save State to Preferences o i command that allows saving the options currently displayed in this window as the default Options Again select the pseudocolor plot type To change the graph s parameters click on the axis labels and select the parameter you wish to view from the menu display Change the X axis to CD14 by clicking on the X axis and selecting Fluor CD14 After changing the Y axis to CD45 the data graph will look like the one on the next page 15 GJ Ungated 931115 BOT Sample O1 fes DAX I Ungated 931115 BOT Sample OT F s a PE k il File Edit Graph Display Go Help File Edit Graph Display Go Help Nojes GE Nojjejsiie HQ CySPE CD45 OrthSc Fluor CD14 PhyEry CD16 Fluor cD 4 I i CySPE CD45 a ey v PEJ Histogram CDF b H Active Gate b b k Statistics Count 10000 10000 100 0 Statistics Count i0000 f 10000 100 096 You may also choose Histogram or CDF cumulative distribution function to create a univariate plot Experiment with different graph types and different options You can also change individual options by selecting them from the Display menu at the top of the graph window Try selecting Use Large Dots under the Display tab for t
11. tables or graphical layouts you will see how fast and easy FlowJo is to use It will take you three to six hours to complete the tutorial you can easily break it up by chapter The tutorial is designed around an example data set The experiments used for the tutorial are based on 3 color immunophenotyping of human peripheral blood mononuclear cells PBMC The steps shown in this tutorial will help you in the analysis of nearly any kind of FACS data The tutorial is only an introduction FlowJo is capable of much that simply can t be covered in an introduction such as this for example there are Analysis Platforms to perform sophisticated DNA Cell Cycle analysis Kinetics analysis Compensation etc You can learn more about FlowJo and in particular how to use these platforms through the on line help facility Whenever you ask for help from FlowJo it launches a web browser and accesses help pages about the topic you selected You can navigate the help pages to find out more about all aspects of FlowJo To access this on line user manual directly point your browser to http www flowjo com v76 en windowstoc html The online help documentation provides tutorials for Compensation Kinetics and DNA Cell Cycle analysis in addition Tree Star provides Demonstration Data to let you explore these platforms http www flowjo com home tutorial html In addition there is a web page for FlowJo FAQs Frequently Asked Questions http
12. want the graphic to appear As shown in the example change the X and Y axes to CD14 and CD45 and ane the graph type to Pseudo color and uncheck smoothing Now click on the Annotate tab near the top of the window This shows a different set of options that control what appears in the graphic see right with Frequencies with Names Show Legend Show Adjunct Histograms C Show Multigraph X Axis Unselect the C Hide Ticks C Hide Numbers C Hide Label checkbox Show a Annotation and Show Gates this will remove the as gates and the annotation text below the graphic Hide Ticks Hide Numbers _ Hide Label Click on OK and the Layout Editor should appear as tate follows UJ FlowJo Layouts AdvWspSAVE wsp File Edit Layout Object View Align Help Kjojsjajajaj ajr Untitled SE off v ForSe 931115 B01 Sample O1 fes Ungated 10000 CysPE CO46 Fluor CD14 FlowJo provides many features of drawing programs such as the ability to align multiple objects To align the two graphs select them both use a marquee selection tool by clicking and dragging to encompass both graphs and then choose the Align menu You may choose to Align or Distribute objects in either dimension For now choose Tops The Layout Editor will reflect your changes as shown on the next page 52 I EYE lowko Layouts Tutorial WS Chapter 6 wip S
13. www flowjo com home fag html Here you may find the answers to a problem you have had FAQs are also instructive you may learn new techniques from reading them You may also have a look at our blog the Daily Dongle for the latest Flow issues or questions http flowjo typepad com Finally we are pleased to be able to frequently update FlowJo to provide new features and analysis capabilities Therefore it is possible that the graphics shovyn in this tutorial may not exactly match the windows that you see when you run the most recent version of FlowJo You can always download the most recent version of FlowJo from the web site listed above Table of Contents DRON OR RR 6 Lesson 1 Workspaces and Basic Data DISPIA Venera 11 L on2 TE aha Ie e E E EE E EEEE 18 Lesson 3 Copying Analyses to Other Samples aaa aaa ennea e eee enee eee e eee ne eee enen eee eee 26 Lesson 4 Groups and Batch Analyses xicccccvcecoemertauseomeatsan eee Hee artes Gd eset wre eee 30 Lesson 5 Modifying NONA Se a ee eee eee ee nee ee ere ee ey een eee eee 36 Lesson 6 Tables and Layouts Collating Data Output aaa aaa aa nnnnna ne eee kene nene rere eee ne ee enet 44 Lesson 7 Creating Simple Graphical Layouts panine rnk Er EEE E EERE EE ERER 50 Lesson Cilin Batch Graphical REPOS errsrrirenerrritre rectis inie EAE EE EEEE AEE EEEE 63 Lesson 9 Generating Complex Batch Analysis pa emanates 70 Lesson 10 Creating Finished IC DOR S pan eriin erii eE EEEE Enk E
14. 279 M2 371 371 M3 1 65 165 To apply them to the group click on the mee T an CD4 T subpopulation and use the ALT v 931115 CO7 Sample 02fes 10000 drag action to move the gates onto the _ Lymphocytes 78 71 7871 Lymphocyte subset of the group Note that Nik ES e her Sh Te Re f f Lymphocytes 56 56 if you were to drop it onto a higher level a w 931115 E07 Sample 04 fes 10000 group name the tree vvould be applied to Lymphocytes 72 64 7261 each sample at the sample level not to the lymphocyte gate That is the gates attach themselves to the row that they are dropped onto JES 34 Your Workspace now shows these gates applied to all of the samples in the group as shown here IE E Fowo File Edit Workspace Groups Tools Windows Help Debug Groups and Analyses 1 Number of Samples T All Samples w GD Lymphocytes we CD td Ay ED MW ED M3 en Naive Name e Statistic Cells_ v 1931115 B07 Sample 01 fes 10000 w ED Lumohorytes FAO a S S ia ia Ww 931115 CO7 Sample 02 fes w GD Lymphocytes wv to CD4 ED Ay ED M2 ED 3 ED Naive w 931115 D07 Sample 03 fcs w GD J ymphocytes w ED CD ST E E 35 Lesson 5 Modifying Group Analyses In this lesson you will learn how to take advantage of previous work you have done dravving gates generating statistics to save time vyhen you do simi
15. 43 Lesson 6 Tables and Layouts Collating Data Output In this lesson you will learn how to transfer any statistical analyses you have requested in the Workspace into other programs You will be able to generate a table of statistics bringing together any set of values from any combination of samples using Groups to define the sample list FlowJo is not a comprehensive data presentation package It provides the tools to analyze and graphically display flow cytometric data Analysis tools such as linear regression very complex graphical displays etc are more suited to programs specifically designed for those purposes However FlowJo does provide tools to collate the output from multiple analyses so that you can import them into spreadsheets tabular data or into drawing programs graphical displays These tools include the Table Editor and the Layout Editor Use your existing Workspace from Lesson 5 or open the tutorial workspace named Tutorial WS Chapter 6 To open the Table NG FlowJo Tables Untitled File Edit Output Help Editor you can click n pa on the fourth button keo teas AL IL JI eaten J 1 Nosi Sample ne in the button E P EH bar or sele ct Untitled A Column Population Statistic Parameter Name the Table Editor from the Windows menu FlowJo shows you the Table Editor window as shown here Click in the title field and type a new name for this table In this window the left side
16. Filename EPIL OpenDocument HTML FlowJo Group All Samples SOL t terate by Sample gt terate by Panel C terate by Keyword Help IL Save LL Apply al i Cancel To export the table choose the menu item Output FlowJo Tables Untitled r Help Cesplay in FlowJo Col D To Clipboard Corl k rizi pe 5 phat To File Cbrl 5 To Printer Cbrl P Untitlec Aubomatic Batch On Simpler UI Corl u The length of the column headings often results in long names as exemplified in the Excel spreadsheet below often much wider than the space allowed for by the columns However you can keep the column names short by giving your own names in the final column of the Table Editor Ox dr amp gt flowjo table xls Compatibility Mode Microsoft Excel non commercial use Trial Ee 53 Home Insert Page Layout Formulas Data Review View c x iE d P si say 7 Te mama AutoSum Arial 10 IA a ES l g Wrap Text General 2 A es p U i E pa E 55 t Fill z d Paste IB ZU rij ri hyr E ajM amp Center 9 lige 00 Conditional Format Cell Insert Delete Format Sot amp Find amp 7 F uE gt 35 E cad Merge iia li ED f Formatting as Table Styles lt 2 Clear Filter Select Clipboard s Font Fa Alignment p Number E Styles Cells Editing A10 SN E C D 7 F G H E 1 Samples Freq of Parent Freq of Parent
17. Groups fools Windows Help Drag this statistic into the aay ea Layout Editor holding the mouse button down Number of Samples as you move the statistic TT CD4 Subsets 4 1 i i CD8 Subsets 4 into the Layout Editor ww i Experiment 1 ee and drop it releasing the E egs Ur PBMC Subsets Fi mouse button to the TT right of the graphic S ey FlowJo creates a text box Statistic Cells that shows you the 931115 B01 Sample 01 fes statistic value as shown T Lymbhorytes in the figure on the next E CySPE CD45 CV age Cy5PE CD45 Mean p 8 8 Freg of Grandparent Y Freg of Parent b Monocytes w 931115 B02 Sample 01 fes w Lymphocytes 5 T cells we DAT Y Freq of Parent w amp CDS T 70 Ll FlowJo Layouts Tutorial WS Chapter 9 wsp File Edit Layout Object Help gjo Ej A a aroo View Align Complex Reprot Complex Reprot Overlay PBMC Subsets PBMC Subsets Batch 1 Scatter Analysis Scatter Analysis Batch 100 Forse 931115 B01 Sample 01 fcs Ungateci 10000 You can add additional annotation to the layout if you need to Here we want to add some additional text to this box To make room make the text box a little bigger by clicking on the lower right handle and resizing it to a larger size Then double click on the text box to have FlowJo open the Text Properties Window as shown below Note that the statistic value has be
18. Help gt FlowJo Reference Manual in the Workspace window The Workspace window is divided into three parts The top part is a button bar The button bar has controls to let you add components to the Workspace like data files statistical analyses etc As you move the mouse over a control button FlowJo displays a description of that control s action in a tool tip The second part of the Workspace above the central divider is a list of sample groups Later you will see how to group sets of samples together for batch analysis The bottom part of the window includes a list of the samples in the Workspace 12 Novy click on the left most button Add Samples You will see a standard Java File dialog navigate to the Advanced Tutorial folder that you downloaded with the tutorial see right vj seme Highlight the folder Experiment 1 and click on the Open button at the bottom to add all of the files in this folder 16 samples one for each staining tube four sets of PBMC with four stains each Your Workspace window should look as shown File name ments FlowJo Tutorials Data advanced Advanced Tutorial Files of type ECS Files La 200903031 Howo Sei Two things have happened NJ Edit Workspace Groups Windows Help Debug FlowJo created a new sample z E group with the same name as the folder containing the FCS Groups and Analyses Number of Samples e I tg Samples Meer of Sap s _ i fil
19. RE fast 1 Subsets and CD8 subsets Again iain aici tatem 1 Select the gate tree and drag it to bring everything into the table Note that you can change the order of the table entries by clicking on any entry and dragging it to another place You can also delete a table entry by selecting it and pressing the delete or key After creating the last table the Table Editor window should look similar to the one shown below PMBC Subset Freq h Column Population Statistic Parameter Name JIT Cell Subsets 1 Lymphocytes T Cells CD8 T Freq of Parent Freq of Parent Freq of Parent Freq of Parent Freq of Parent Freq of Parent Notice in that picture how the column names were assigned for each of the columns generated in the CD8 Subsets table These names will appear both within FlowJo s tables and when the data is exported to other applications E FONTO Tables CDE Subsets File Edit Output Help Honn Sample v Pat Column Population Statistic Parameter Name 1 Lymphocytes CD8 T M3 Freg of Parent Freq of Parent PMBC Subset Freq T Cel Subsets iymphocytes CD8 TiNaive Freg of Parent Regdpaat 1 ae loam O Lymphocytes cO8 TIN Freq ofPaent at You can now apply these table a Lymphocytes COSTIME Medan GEO I nj templates to the appropriate groups and get output tables with specific sets of statistics just select any group gt ia ka Se pes
20. You will be able to generate layouts with multiple different graphics text items lines etc and you will learn how to create overlays of graphs In Chapter 8 you will learn how to create Batch Reports where the layout is applied to all of the samples in the workspace Finally in Chapter 9 you will learn a few of the features of the Layout Editor that let you create complex multi sample layouts including statistics and graphs from multiple tubes in a panel This lesson continues with the Workspace document you have finished from Chapter 6 alternatively you can open the workspace named Tutorial WS Chapter 7 f To open the Layout Editor either select Layout Editor from the Windows menu T press Ctrl L or click on the Layout icon in the row of icons at the top left of the Workspace window You will be shown an empty layout editor In the Layout E d itor eee window the left portion of File Edit Layout Object View Align Help the window is a list of all of KoNNa Ja DOSE the layouts you have created for this workspace lis etted Untitled A You can have as many layouts as you wish For example in the Demonstration Workspace shown in the introductory chapter there were Layouts for generating patient reports for verifying scatter gates etc You can name the layout by clicking in the name field Untitled 1 near E the top of the window and Forse typing a new name 931115 B01 Sample 01 fts Ungated
21. all of the handles in to make this gate smaller Ungated 931115 B01 Sample OI fes File Edit Graph Display Go Help post S SGX Click the Workspace window it will now reflect this modification by drawing the newly modified lymphocyte gate in black See the second line in the figure below T sch5 wspflowo paa Bie Edt Workspace Groups Tous Windows Help Debug I Groups and Analyses umber of Samples 100 IT All Samples I i s m la 3 b T CO4 Subsets d Forse b ic CDS Subsets i T Lot Experiment 1 16 b Options LF PBMC Subsets 4 wo Lymphocytes b Active Gate E cose a CySPE cv Statistics Count 10000 10000 100 0 CySPE Median a E Freq of Grandparent e Statistic Celk w o 931115 B01 Sample 01 fes 10000 dd Lymphocytes 69 00 6900 w ED Cr A Sd GJ ICYSPE CD45 CV 34 68 Cy SPE CD45 Median 473 99 E Freq of Grandparent 62 54 E Freq of Parent 90 d How can we update all of the samples is 7005 1085 for this experiment All of the samples Cham fass Os for this experiment belong to the group v wostiizeon sammie st ts E Experiment 1 the group that was Tite nn Om created when you dropped in the folder T Po mae of FCS files So you can use this group EST iiai a 2 for analyses that apply to all samples Sree eee on Ho Alternatively you could create a new 7 1115B0 sampe ots een group select all samples and drag
22. dataset 1 originalDataset 1 name CYT gt skey datasets choose Line Weight No Line When you are 1 originalDataset 1 name f SY S gt E Fatient ID Key dataset 1 originalDataset 1 name Cells finished click ON OK Insert Keyword none gt vi In the Layout Editor magnify the view to Ee E 100 The Layout Editor should look something like that below If you wished to have a particular keyword value in a different font or color you would have to make a Font Seri e coo NE separate text box for it and format it Size 12 m Justify center gt Style bold e accordingly Fil Color J Line Weight EC Line Color E Line Style Solid Ka The next job is to add plots into the middle I Resize box to fit text panel graphics that shovy hovy each antibody staining panel was gated This is similar to fse se eet La Lew some of the layouts you created in previous chapters First drag the parent sample 931115 B01 Sample01 fcs from the Workspace into the Layout Editor dropping it over the Gate Settings panel You will note that you cannot see the text because the graphic text is black We will change this totara B J FlowJo Layouts Tutorial WS Chapter 10 wsp File Edit Layout Object View Align Help Mosaea de Itjej l Untitled 1 Cells ID 1001 gjej ftube J Complex Reprot Complex Reprot Batch 1 Overlay PBMC Subsets PBMC Subsets Batch 1 Scatter Analy
23. holds the list of existing table templates The right side will show the items in the currently selected template What is a table template When you define a table you will add rows to a table template Each row in the template defines one statistic that you want to export such as frequency mean fluorescence of FITC etc When you create the table FlowJo cycles through each sample in the current group and requests the particular statistic defined in the table 44 NOTE When you create the table and import it into another program you will have one row in the table for every sample in the current group and one column for every statistic in the table template Rows in the template create columns in the table You may define as many table templates as you wish you could use one for each different set of statistics This one as you may have guessed will be used for generating a table of the major PBMC subsets analyzed in the first stain combination Note that you can apply a template to any group it doesn t matter which group was active at the time that you defined the table template However most likely your table will only be applicable to one group of samples Go back to your Workspace and ES select the PBMC subsets group From the first sample only select Untitled Population Statistic the L y m p h OC y t es and C D 4 5 Gi 1 Lymphocytes Freq of Paren Lymphocytes CD45 Freq ol aren subpopulations and then t
24. them TE o ser T to the new group to add them and then de H use that group Drag the modified w T7 931115808 Sampie 01 fcs 10000 lymphocyte gate to the Experiment 1 T aaa ee ran group Now look at the Workspace Om age shown on the next page Dm z ot FL 931115 C01 Sample OF fes 10000 w CO Lymphocytes T Qi ea w ED cogs TG TI i677 Cy SPE CD45 CV 34 05 Ka LYaPE un Med 176 75 40 T U Tutorial WS Chapter 5 wsp FlowJo mm ol File Edit Workspace Groups Tools Windows Help ujku ose Number of Samples T All Samples Now all Lymphocyte subsets are drawn in ii CD4 Subsets 4 E bold blue text denoting that they are gt iympioces identical to the group s version Open any J sample s graph to verify that it has the right ay E Statistie Cells wi 931115 B01 Sample 01 fes p 650 Lymphocytes Monocytes wi 931115 B02 sample 01 fes w ED Lymphocytes w ED T cells w CD CDA T Freq of Parent w CD CDE T gt Freq of Parent w Double Negative T Y Freq of Parent wi 931115 B07 sample 01 fes w 65 Lymphocytes b amp CD4T wi 931115 B08 sample 01 fes p 60 Lymphocytes wi 931115 C01 Sample 02 fes p 60 Lymphocytes Monocytes wi 931115 C02 Sample 02 fes w ED Lymphocytes w T cells w CD CDA T gt Freq of Parent w CD CDET lymphocyte gate One final lesson before moving on to tabular and graphical present
25. view the graphs To view a complete report just click on the batch generation button Set the geometry to 2 columns and generate a new Layout FlowJo now co iterates only four times and 1660 931115 bos Sample 01 fos cosT oe generates four tiles one for each unique value of Cells Set File gt Avoid Page Breaks You should see a window such as the one below Forse Phy Bry CD45RA I Phy Ery COGSSRA Fluor L selectin FlowJo Layouts Tutorial WS Chapter 9 wsp File Edit Layout View Align Help SJ OjN 8 4 2 qer l l Na a You may wish to experiment Complex Reprot N E ar T Cell Analysis i I l l J some more by dragging in the Complex Reprot Batch 1 pray ge ce ea e folder for Experiment 2 on to the PBMC Subsets paycswsos Baicet gamse f A T mod EP current workspace and Scatter Analysis pi Pa gt i 2 b a regenerate these layouts They will be considerably more complex as you will have 18 patient samples however you will quickly appreciate how little additional work is involved in your future analyses T Cell Analysis T Cell Analysis In the next Chapter you will learn some additional Layout Editor techniques including creating Grids and placing background graphics underneath the reports 76 Lesson 10 Creating Finished Reports In this chapter you will learn how to use some of the advanced features of FlowJo s Lay
26. views of the subset hierarchy much like you can do in the explorer window In the genealogical terminology that FlowJo uses the CD4 T CD8 T and Double Negative T subpopulations are siblings and are children of the T cells subpopulation grand children of the Lymphocytes subpopulation etc Remember that the frequencies and cell counts shown to the right are with respect to the parent sample The Workspace window below reflects this hierarchy PhyEny CD8 P Options k Active Gate Statistics Count 4745 10000 47 45 Li 20090303 T FlowJo File Edit Workspace Groups Tools Windows Help Debug u 07 29 E e Groups and Analyses Number of Samples lt u All Samples 16 u Experiment 1 16 E Name dd Statistic Cells CySPE reagent Fluor reagent PhyEry reagent NY 931115 B01 Sample 01 fes 10000 El CD CD3 CDE Fi H 931115 B02 Sample 01 fes 10000 w 2 Lymphocytes 78 22 7822 w 0 T Cells 47 45 4745 Gi CD4T 14 50 1450 Gi CD8T 22 25 2225 2 Double Negative T 9 40 940 U 931115 Bo7 sample 01 fes 10000 L selectin CD45RA j 931115 B08 Sample 01 feces 10000 L selectin CDO45RA 931115 CO1 Sample 02 fes 10000 CD14 CD16 931115 C02 Sample 02 fcs 10000 CD CDE 931115 C07 Sample 02 fes 10000 L selectin CD45RA 931115 C08 Sample 02 fces L selectin CD45RA4 Add the Freq of Parent statistic to each subset You may also add it to the first subset CD4 T us
27. 000 10000 10000 10000 10000 10000 Name Statistic Cells CySPE reagent Fluor reagent PhyEry reagent 10000 CD45 cD14 CD16 CD3 L selectin L selectin CD14 CD3 L selectin L selectin CD14 CD3 L selectin L selectin L selectin L selectin first line in the sample list you will see the graph shown below Ungated 931115 B01 Sample OT Fest HONE File Edit Graph Display Go Help Yoda gate T This is a Graph Window A Graph window will show you a plot of the data There are several different kinds of plots that can be used to display the data The default plot that you see first is determined by the Preferences setting To change the Graph click on the Options disclosure triangle at the bottom of the Graph window 50 100 150 200 Forse Y IT k Options b Active Gate F Statistics Count 10000 10000 100 0 This button brings up the options shown to the right By clicking on the graph type drop down menu you can select among different types of graph plots Select contour to see the graph displayed below Options Graph Type Eea ea j I Smooth a k Active Gate kk Statistics Count 10000 10000 100 0 200 Ti If you click on the menu Edit gt Save State to 8 Preferences this plot will become the default Sach Tyee ET e 5 type of plot to be opened Note that this _ Cae
28. 10000 To add a plot to the Layout click on any subset in the vvorkspace and drag it into the layout view For now click on the top level ungated sample 931115 B01 and drag it into the Layout View You will immediately see a graphic corresponding to the subset The default graph that is shown is the same as how the subset was last viewed In addition FlowJo creates an Annotation text box below the graphic that contains some pertinent information To view gates double click on the graph this brings of up the Graph Definitions dialog box select the Annotate tab click in the Show Gates box you should see a check mark when selected and click ok at the bottom of the dialog box 50 Any graphic item can be resized or Jl FlowJo Layouts AdvVVspSAVE wsp moved just by clicking and Elle Edit Layout Object View Align Help dragging To resize click and drag ao JN at 4 a aos on one of the four handles at each corner You can also change the magnification of the view by clicking on the Scale popup menu near the top of the window see example right One of the important aspects of the Layout Editor is that it is live This means that any time you change or move a gate or modify an analysis in the Graph Window FlowJo will automatically update the Layout Editor if needed Thus you can use the Layout Editor to provide instantaneous feedback for gating operations where you can DE Un
29. 55 cD CDT 14 43 1443 Y Freq of Parent 30 35 w c CDBT 22 39 Freq of Parent 47 09 w Double Negative T 9 53 Y Freq of Parent 20 04 w 1 931115 002 Sample 02 fes w amp Lymphocytes 79 42 w GD T Cells we CDIAT Y Freq of Parent v SCD8T i m 32 Now we move on to the next set of analyses the N 2009030341 FloviJo CD4 and CD8 subsets Again we will use groups Jete Edt workspace Groups Tools Windows Help Debug to speed up the process and ensure that MGA ea E identical gates are used for common gatings like Lymphocytes While the CD4 and CD8 subset ___swuos and nase Luna analyses used different stains vve can still copy v iT Goi Subsets I the lymphocyte gate since it is based only on SE ii CDS Subsets Forward and Side scatter Click on the T Experiment 1 Lymphocyte gate from one of the samples e g 7 the first one in the currently selected group and w Lymphocytes drop it on top of the CD4 subset group The gate Name a J Statiste Jace is now applied to all of the CD4 subset samples a a nes w amp T Cells 47 45 4745 w amp ECDAT 14 50 1450 Y Freq of Parent 30 56 v amp CD8T 22 25 2225 Y Freq of Parent 46 89 wY amp Double Negative T 9 40 940 Freq of Parent 19 81 931115 C02 sample 02 fes 65 Lymphocytes 78 91 w c T Calls 57 95 v amp CD4T 16 97 Freq of Parent 29 28 w amp CDBT 37 69 Freq of Parent 65 04 w 659 Double Negative T 1
30. 87 Se Gina af Drin E NI LAJ 20090303 T FlowJo File Edit Workspace Groups Tools Windows Help Debug T 0 zt 8 S Groups and Analyses Number of Samples T All Samples 16 U D Lymphocytes T CD8 Subsets T Experiment 1 E PBMC Subsets w i T Cell Analysis w amp Lymphocytes Name 2 saute sc When you click on the CD4 subset group w 1 931115 B07 Sample 01fcs 10000 Lymphocytes 7770 770 you will see the four samples in the lower 10000 yee EE r Sample panel with the lymphocyte gate U 931115 D07 S le 03 f 10000 i b b w Bat TS DOT Sample 03 fes oe E now drawn in green italic as shown on the w 931115 E07 Sample 04 fcs 10000 left d Lymphocytes 72 61 7261 i Eile Edit Graph Display Go Help 5 Ke Ba elboj Lymphocytes N 120 T Double click on the first sample s lymphocyte at subset to open its graph and change the graph to show a histogram of CD4 Using the range gate tool create a gate for the CD4 T cells like the one 3 9 shown right T Click on the Down arrow in the box at the upper right corner of the Graph Window in order to l view the population defined by the active gate RE Now you are looking at the CD4 subset within the lymphocyte gate a p Options Active Gate CD4 CD4 15 61 ST AN 19090 e k Statistics To show the graph on the left but without gates click on Options and select Contour as the graph type an
31. D45 Lymphocytes is 114 00 E Freq of Parent 89 86 lt H 931115 B02 Sample 01 fes 10000 CD3 CDS the distribution of CD45 has a U 931115 BO7 Sample Us 10000 L selectin GDASRA Coefficient of Variation of U 931115 B08 Sample 01 fes 10000 L selectin CD45RA U 931115 C01 Sample 024es 10000 CD14 CD16 35 2790 The Freq of Parent U 931115 C02 Sample 02 fes 10000 CD3 CDE statistic tells you the frequency ui UU C07 Sample 02 fcs 10000 L selectin CD45RA il of CD45 events within the parent subset Lymphocytes You will note that the value 89 86 is equal to 63 80 the frequency of this subset within the sample divided by 71 0 the frequency of lymphocytes within the sample The Freq of Grandparent shows you the frequency of this subset within the population two levels up in this case the sample itself Thus for this subset the Freq of Grandparent is the same as the frequency of this subset within the sample These statistics are listed below the CD45 subset to denote that they are statistics for that subset only jj Next you will create another gate on the Fle Edt Graph Display Go Help JI sample this time for monocytes Double click oO o al Aja djejpjejj in the Workspace to open the graph for the sae lt gt first sample choosing the Forward vs Orthagonal scatter display option Draw a gate around the upper population as shown to the left and name this subset Monocytes Note that the currently selecte
32. E Szen LI selecting them and pressing the delete e ae w amp Double Negative T 187 key Select the lymphocyte amen e u 931115 CO7 Sample 02 fcs L selectin CD45RA subpopulation that you just created U 931115 C08 Sample 02 fes L selectin CD4SRA u 5 Sample 03 fcs and press the delete key When you SHE Habe Sub Shia aia delete the lymphocyte gate all of the U Fat 115 POE Sampla gates lsali CDRA subsets of lymphocytes will also e H 931115 E02 Sample 04 fes cpa CD8 deleted this is because those subsets have no meaning without a lymphocyte gate being 28 present Remember every subset that you name is in reality a subset defined by all of the gates of its ancestors the parent gate grandparent gate etc VVhen you delete a gate FlowJo lets you know that all of its descendants will also be deleted in the confirmation request r Confirmation Are you sure you want to delete the population Lymphocytes I Don t show this message again One more detail when you create a subset the gate used to create that subpopulation remembers both the parameters and the stains on which the gate was drawn In other words the CD4 and CD8 T cell gates drawn above were created on a sample that had PE CD8 and Cy5PE CD4 For this and other reasons it is important that you carefully enter the appropriate stain names when you collect the samples Naming stains appropriately is good practice anyway you will then always have proper
33. Experiment 1 1 PBMC Subsets w 7 T Cell Analysis Name ai Statistic Cells 931115 B08 Sample 01 fes 10000 w amp Lymphocytes 744 H 931115 C08 Sample 02 fcs 10000 w amp Lymphocytes 7752 t CD8T 0 931115 D08 Sample 03 fes 10000 Ww Ei Lymphocytes 7204 e5 CD8T 0 Ww 931115 E08 Sample 04 fts 10000 w cD Lymphocytes 7106 e5 CD8T g Watch the open Graph window as you do this the four gates will appear in this window as they are attached to the sample Eile Edit Graph Display Go Help XJoj Joj Alas PhyEn CD45RA from one of the samples see left Drag this set Fluor L selectin V of gates and drop it onto the CD8 T gate in the 975 CD8 subset analysis group k Active Gate k Statistics Count 2470 10000 24 790 37 Lymphocytes CIS T 931115 B08 Sampl m Ele Edit Graph Display Go Help Lyn u PhyEry CD45RA 4 k Options k Active Gate amp Statistics Count 2470 f 10000 24 7 Move this window off to the side as well and watch it during the next operation In the Workspace window you will note that the subpopulations for the first sample are drawn in black this is because you modified them and they are no longer equivalent to the versions in the group Molalla IIg It is apparent that the gates are not positioned properly for CD8 T cells these are the gates that were appropriate for CD4 subsets In particular th
34. FlowJo Data Analysis Software for Flow Cytometry Advanced Tutorial STAR FlowJo was written by Adam Treister and Mario Roederer beginning in 1996 based on concepts developed at the Herzenberg laboratory at Stanford We are indebted to our active and enthusiastic users worldwide for their ideas discussions and tireless testing of new versions FlowJo its tutorials documentation and web site are Copyright Tree Star Inc 1997 2010 All Rights Reserved e FlowJo Advanced Tutorial e MMX Revision Date February 22 2010 Version 7 6 FlowJo Tutorial FlowJo is a software application designed to create an integrated environment for viewing and analyzing flow cytometric data This environment is presented in the form of a Workspace The Workspace contains a list of all of the data samples that you load the gates statistics and other analyses that you apply and the table templates and graphical layout templates that you design The Workspace is saved as a FlowJo document on your hard disk when you re open the document you will see the status of your analyses as they were when you last saved the Workspace This tutorial is designed to introduce you to the program Reading through it you will learn how to operate FlowJo Run the program as you perform the steps in the tutorial so that you can get the best feel for how the program works As you watch FlowJo perform various operations such as creating new graphs statistics
35. Freq of Grandparent Freq of Parent Freq of Parent Freq of Parent Freq of Parent 2 931115 B01 Sample 01 fcs 69 00 91 17 62 91 91 17 21 00 47 00 43 67 3 931115 C01 Sample 02 fcs 77 57 96 9690 75 21 96 96 14 5890 39 51 47 46 4 931115 D01 Sample 03 fcs 53 54 92 67 49 72 92 87 31 30 54 0690 40 1396 5 931115 E01 Sample 04 fcs 54 68 91 77 50 18 91 77 33 37 49 36 45 07 6 Mean 63 70 93 19 59 51 93 19 25 0690 47 48 44 08 7 Standard Deviation 11 62 2 61 12 12 2 61 8 84 6 07 3 06 8 M4 gt PMBC Subset Freq Si DO Ready 100 47 The Table window is always current When you create a table FlowJo goes through all the samples and makes sure they are recalculated according to the latest modifications If you now go back and change the lymphocyte gate apply that change to all of the samples then that change will be immediately reflected in the table window From the Table Editor you can create new table templates duplicate existing tables or delete existing table templates using the buttons across the top parece cos care spa Click on the Plus button and name Z l the new table T Cell Subsets H From the Workspace select the T Cell Analysis group Select the gate tree and drag it from the first sample into the Table Definition tc Can pes FE ass Window you will see all 8 rows IlymphocytesjT Cells CD4 T Freq of Parent Freq of Parent Create two more tables for the CD4 a
36. Ge Ma E gt Naive w 931115 B08 Sample 01 fcs ow ED Lymphocytes r ED Wt E GySPE CDA Mediar Freq of Parent w ED M7 E CySP5 CDS Mediar Freg of Parent r GA Tutorial WS Chapter 9 wsp FlowJo File Edit Werkspace Groups Tools Windows Help mmga Groups and Analyses Number of Samples Statistic 68 51 6851 91 36 6259 47 56 T73 88 62 59 oo 22 21 15 2175 10000 FAQA r4or 62 25 4670 30 28 1414 30 12 46 72 2182 40 72 20 34 950 20 34 10000 For Taar 16 4 4 16 4 4 Cels 10000 a ZEF d o 28 154 39 94 b 10000 Fear roar 33 76 atz 73 18 33 18 13 36 IZ Qa 22 13 350 QI FlowJo Layouts Tutorial WS Chapter 9 wsp 3 Select the annotation text boxes Fie Edit Layout Object View Alban Help under the three newest graphs and Scatter Analysis Batch JE IPS LIES E E QO bel press the delete key to remove the e ae annotations Move the graphs a T Cell Analysis little closer together Novy you have smana o AS _ created a graphical report that draws graphs from different tubes D of the same patient Now set up the Lymphocytes are 8922 E 10 iteration by selecting Cell as the ONE iterator and tube as the e SAN discriminator as shown to the left ta Change the patient ID to ID 1004 All four graphs and the statistic change and now are drawn from the fourth patient sample in the vvorkspace You can select any patient sample to
37. Layouts tutorial WS Chapter 9 wsp Fie Edt Layout Object View Align Help Debug SCIEN Gje tjel Complex Report l Scatter Analysis Batch Untitied 1 Untited 1 Batch Lymphocytes are 89 22 of total events 931115 B01 Sample 01 fes Ungated 10000 Make the Layout Editor Window larger by dragging the lower right hand resize the window in order to make room for more graphs in the Layout View Back in the Workspace Window click on the CD4 T subset and control click the CD8 T subset under the next two samples as shown to the right Click on one of these and drag both will be dragged into the Layout View just below the first graphic Now two more graphs appear Adjust them as shown next 15 Oo O i a ICAEN Off ki Cell Analysis CySPE CD4 931115 B02 Sample 01165 T calls 4658 corner to Al Samples ir CD4 Subsets E COO Subsets Experiment 1 ED Lymphocytes tur PBMC Subsets 0 T Cel Anatysis Mame lt w oa 931115 BO1 Sample 01 fcs w 55 Lymphocytes w D coqs 2 CySPE CD45 CW CySPE CD45 Medan Freg of Grandparent t Freg of Parent gt Monocytes w i 931115 B02 Sample 01 fes 55 Lymphocytes w 65 T Cells FTE ODA E Freq of Parent w amp coaT E Freq of Parent Double Negative T Freq of Parent U 931115 B07 Sample 01 fcs ED Lamohocytes i ED NM Ex A
38. R4 Y t Ljmphocytes Fai bo 469 E Cite T 30 54 2257 Mi 19 15 1430 iE Mi Ft 567 i MT 3872 209 t Mahe 25 43 1899 1 931115 CO8 Sample 02 fes 10000 COGE Les l tlin CD45RA Lymphocytes 75 68 7508 T e T 46 97 7550 Mi 20 63 1567 M2 9 84 ras D MI AR AT tin You are now finished with the T cell analyses we will now go on to the PBMC subset group Alt click and then drag first the Lymphocyte then the Monocyte gates from the first sample in Experiment 1 to the PBMC Subsets group This applies the analyses to the entire group see left Now if you click on the Experiment 1 group you can look at all of the samples from this experiment in their full glory left Note that the assignment of color and style to the groups helps you identify which group any particular gate comes from 39 As a final tune up for the gating analysis you will modify the lymphocyte gate and update all of the samples for this experiment to have the new version of the gate It is important to see how the program s dynamic recalculation can be used to modify either single gates or gates shared among multiple samples Open the graph of the very first sample at the top level in the sample list double click on the top line You will see the two gates the lymphocyte gate and monocyte gate Select the lymphocyte gate and drag the individual handles so that it is tighter around the population see the graph on the left Move
39. Scatter Analysis Orhsc sets of gates and statistics then the layout may not operate as desired on other groups vyhich don t have those gates Once again if you select Off for the current iteration value then FlowJo shows only the original graphs for this layout Vievy the ones that you dragged and dropped into the view irrespective of what the current group is Understanding how FlowJo generates a graph for any given Layout item during batch processing is very important FlowJo draws a graph in the Layout Editor when all of the following criteria are met 1 the current sample i e the current value of the Iterator has the parameters that are displayed in the graph like Forward Scatter FL3 FL4 etc and 2 the current sample has a gating tree that has exactly the same subset as what is desired in the graph i e if the graph was dragged from a CD4 subset of Lymphocytes then FlowJo looks for a CD4 subset of Lymphocytes in the current sample So far you have only looked at the Layout Report for individual samples one by one To look at graphs for all samples at once click on the Batch button above the Iteration Popup menu This dialog will let you set the options governing how the report will look You can set the format of the report the group that will be processed the geometry of the rows and columns in the report and other options to set FlowJo to perform the correct operation without asking you eve
40. T EE EERE n Ei E Erkin rik TI Introduction This chapter is designed only to give you a quick overview of the powerful batch features that FlowJo provides don t worry about trying to learn how to use the program during this demonstration Chapters 1 through 9 are designed to teach you details of using FlowJo For this demonstration you will load a sample data set into a previously designed Workspace that already has gates statistics and graphical reports designed You can think of this workspace as a Template which could be used over and over for similar sets of experiments To begin this tutorial first locate the Advanced Tutorial Data and Workspaces Folder on your PC either copied from the CD ROM that came with FlowJo or downloaded from the FlowJo web site Open this folder you will see a number of Workspace documents wsp and a folder containing the Tutorial data inside it has 2 folders of experimental data collected on different dates Locate the workspace document named Demo_Workspace wspt and double click it to launch FlowJo The unlicensed version of FlowJo can only read demonstration data Kaloj Biases ist from Tree Star Inc You can run this tutorial or look at special FCS e an files that we provide but it will not About Dongles to view our trouble shooting read data from your lab until you v have obtained a serial number or dongle from Tree Star Serial z s If you have just purchased FlowJo and n
41. T SCOPE CDB Media 7398 0 same name Then every sample that has w 9911 15 606 Sample 02 fes n the group s version of those analyses Ter 3750 will also get the new version that you TE CSPE CD Median 7478 have copied to the group _2 Freq of Parent 41 15 cD M2 6 02 Y CySPE CD8 Mediar 75 82 Freq of Parent 16 05 S M3 6 06 CAPE CD8 Mediar 71 93 gt Freq of Parent 16 16 v 9 Naive 984 f CySPE CD8 Mediar 72 39 2 a a fe 24 4 w 931115 D08 Sample 03 fes v E Lymphocytes 69 73 v CDBT 22 37 w D U 141 Confirmation The node 931115 CO8 Sample 02 1cs already has a Child with the same name Do you want to replace it Don t Ask Again If you choose to Synchronize Group s Gates FlowJo will automatically update the gates for all the samples in a group as soon as you adjust a gate on one sample This option saves time by applying newly adjusted gates automatically to all the samples in the group skipping the step of dragging the newly adjusted gate up onto the group name However it does not allow between sample variation of gates All the gates will be the same as the last adjusted gate on any sample in the group aj Modify Group Appearance Name CD4 Subsets Color Group Name N Style italic KA Sample Inclusion Criteria Live group Include samples that use the Following staining CDI4 CD16 CD45 CD3 CO8 CD4 L selectin CD45RA CD4 L selectin CD45R 4 CDS
42. ame this way no confusion can arise You can however give the same name to gates under different subpopulations Later you will see examples of how this is useful to more complex analyses FlowJo identifies subsets using the names of populations When you later create graphical layouts or tables you tell FlowJo that you want information about a population like CD45 Lymphocytes FlowJo will look within all of your samples for a subset or statistic with this name and ancestry to extract the information you want Importantly the precise Lymphocyte or CD45 gate can be different between different samples but FlowJo will still recognize the populations and get the data you want from them In fact you can even draw a gate on completely different parameters but if you name it Lymphocytes and it is at the sample level then FlowJo assumes you have selected the same kind of cells as every other Lymphocytes gate attached at the sample level In conclusion The name of a subpopulation is the fundamental name by which FlowJo identifies subsets of cells 25 Lesson 3 Copying Analyses to Other Samples One of the basic principles of FlowJo is that virtually all analyses that you perform on a population of cells a sample or a subset can be applied to other populations using the drag and drop feature Hence when you click on an analysis for instance a statistic or a gate creating a subset and you drag it to another place in the Workspace
43. ample Workspace you created and change the graph to Forward vs Orthagonal scatter and the graph type to contour You will now create a lymphocyte gate with the I polygon tool draw a new gate around the lower population as shown on the 200 right vi T E Options AS SOON as you close the polygon FlowJo k Active Gate Lymphocytes Lymphocytes 1 00 asks you to name the subset that you have statistics Count 10000 10000 100 0 just gated see below FlowJo provides a default name however you should choose a name that describes the population that you gated Names are very important within FlowJo analyses The success of your future analyses depends on having analyses with descriptive names For example in this case you have gated lymphocytes so name this subset Lymphocytes The Help button will provide more information about naming subsets Type the name into the box or choose from the pull down menu You can add names you type to a list of favorites by clicking on the plus icon If you click on the Display button FlowJo then creates the gate and opens a new Graph window showing only the events contained within the gate you just drew For now just click on the OK button Subpopulation Identification You will note some changes to the Graph window The active gate line second line from Enter the name of this subpopulation ee the bottom has the name of the currently a selected gate and the fraction o
44. ample 01 fcs 10000 CD4 CD3 cD8 a i wv 69 Lymphocytes 79 12 7912 defined for this group You can v T Cells 47 55 4755 see all of these in the example i oa i v DCDST 22 39 2239 vvorkspace on the right ET s c w 69 Double Negative T 9 53 953 Y Freq of Parent 20 04 w j 931115 C02 Sample 02 fes w amp Lymphocytes 79 42 w amp T Cells 58 16 wv amp cD4T 16 92 Y Freq of Parent 29 09 w cpdstT 37 76 The color style annotation of the analyses is an important cue for you to note Any analysis that appears in the Workspace in the color and style of the group is guaranteed to be exactly the same as the group s version Thus the gate will be in exactly the same location applied to the same parameters etc This is how you can guarantee that the Peer Te em ner m exact same analyses have been done on all e cai EPEAT ITE of your samples We will see later how to 3 OP 2 Ba E modify analyses for particular samples ee ee ee __Nunberofsamles_ Use the menu item Workspace gt Edit T All Samples 16 la T CD4 Subsets 4 I Columns to remove the reagents from T CD8 Subsets 4 _ fe eee the workspace to simplify our view You 7 PBMC Subsets 4 can choose to display or hide whatever w ut T Cell Analysis 4 N w Lymphocytes attributes you prefer w Ci T Cells Name dh Statistic cels w ij 9391115 B02 Sample 01 fcs 10000 5 Lymphocytes 79 12 7912 w i T Cells 47 55 47
45. ations merging analysis trees Let s assume that you modify an existing analysis tree on a sample for instance to add several statistics to different subsets You would now like to propagate these changes to the whole group or other samples without having to drag each new statistic individually FlowJo gives you the ability to easily merge analysis trees that are very similar adding only the new or updated analyses to the destination When you drag an entire analysis tree onto a population that already has parts of that tree FlowJo assumes that you want to replace all of the existing analyses that have the same name As an example select the CD8 Subsets group and add two statistics to the first M1 subset Click on the M1 subset and click on the Add Statistic button Ask for the Median of Cy5PE CD8 and the Frequency of Parent statistic Then shift click these two rows in the Workspace and drag them to each of the M2 M3 and Naive populations 4 Here is an example where you have lek modified the existing tree in sample SS B08 and wish to add the changes the 2 VI E E statistics to C08 You could simply drag trasama 0 E g the two statistics nodes all the way to the mmmnmummmmn M1 subset with C08 resulting in the Ter vvorkspace shovyn on the right Then Ms you could continue this process 5 Experiment 3 e k 3 PBMC Subse However this is tedious especially if v di TCell Anatysis
46. ayouts Tutorial WS Chapter 7 vysp File Edit Layout Object View Align Help SX O N a A a a Overlay Scatter Analysis de AENA Overlay off v H Population Name I Lymohocvtes l Fluor CD14 Cy5PE CD45 Once you create an overlay graphic you can easily change its appearance axes and plot style just like any other graphic mi Population Name Monocytes IELymphocytes Graph Definition si Double click on the item you will be shown the 131115 BO1 Sample D1 fcs Graph Definition window Change your graph to f meem show ForSc vs OrthSc forward vs side scatter X Axis Forse M vo Graph Type Dot Plot vi Contour Levels Now the Layout Editor Window should look Foreground E something like the image below See that the dot Background clusters reflect the gates which defined their colors Controls Checked items remain locked during iteration Double click to change setting 931115 B01 Sample 01 fes Monocytes 931115 B01 Sample 01 fes Lymphocytes FlowJo Layouts Tutorial WS Chapter 7 vysp File Edit Layout Object View Align Help vojnjejajaj aji ne Scale Horizontal 100 Vertical 100 t zl l Overlay KA i BO E Lymphoctes Monocytes Poe Double click on the graphic again and choose to display a histogram of CD16 FlowJo now shows you a histogram overlay like that shown below FlowJo Layouts Tutorial WS Chapter 7 wsp File E
47. catter Analysis 10 ysPE CD45 931115 B01 Sample 01 fes Lymphocytes Tai Re 56 Double click on the new plot The Graph Definition EE window will appear as shown left Change the graph Pere Larte ors specification to be Dot Plot of CD45 vs CD14 X Axis CySPE CD45 Graph Definition Click on OK and the Layout View now displays a dot plot as shown below Graph Type Dot Plot Contour Levels fe FlowJo Layouts 4march09_wsp File Edit Layout Object View Align Help Debug Joao ej de MODENA off Foreground Background Controls Checked items remain locked during iteration Double click to change setting 931115 BO1 Sample 01 fes Lymphocytes Q o Oo u Scale Horizontal 100 86 Vertical 100 931115 B01 Sample O1 fes CYSPE CD45 mpir ia Any graphic item can be made into an overlay by dragging any other subset and dropping it onto the overlay FlowJo Layouts Tutorial WS Chapter 7 wsp File Edit Layout Object View Align Help X O A a Q 100 vaj GJGJBEJM t iej l Overlay off v You can overlay different subsets from the Saige TE same sample or overlay plots from different Demas samples For now select the Monocytes JI subset from the same sample and drop it on the graphic the sample name will appear in the legend to show that is was dropped You should now see the mul
48. cell cycle analyses and calibration standards Everything you do is recorded and saved so that you don t have to redo the same steps To generate one of these graphical reports you need to open E the Layout Editor Window You can either select this from the fle Edit Workspace Groups Tools Windows menu or click on the Layout icon in the Workspace DET pay ee G lt ax _ ea window as shown at right Gi Jups and A iu AN Samples p tur CO4 subsets FlowJo now shows you the Layout Editor window opening one of the four graphical layouts that have been designed for this workspace see graphic on the next page In the left hand portion of the Layout Editor window you will see a list of different layout templates To fully see the Abutting Graph you need to click on the CD4 subsets group in the Workspace For the Overlay example choose either the All Samples or the Experiment 1 group and select one of the menu entries showing the Patient ID on the top right near Cells Finally click on the Layout named Scatter Gates FlowJo Layouts Demo Workspace wspt File Edit Layout Object View Align Help Debug One of the important first steps in analyzing an experiment is to make sure that all of the scatter Ixjojsjejajaj Qjioos gates are appropriate for each TT EE scatter Gates sample You will use this layout to s rsge 5 verify the scatter gates for all 16 overiay came tubes collected i
49. ces With reference to samples in another group C Show all keywords in menus Only choose fram d samples in Group No specified group t Also include all Clase 30 stains present in the vyhole list of samples They are presented in channel order in this case FITC PE Cy5PE Stains which were left blank at the time of collection would be noted with an ellipsis Click on the CD3 CD8 CD4 combination telling FlowJo to select all samples with this combination Enter the name T Cell Analysis in the box at the top and click on the Create Group button The window is still present you will notice a new line in the Workspace window corresponding to the group you just created You will now create your second group corresponding to the analysis of the CD14 CD16 CD45 stains Again click on the stain combination and enter the name for this group PBMC Subsets Before you create this group change the color to red click on the color box and select Bold Italic from the Font Style popup menu Your Create Group window should look as shown at the right Now click the Create Group button to create this group Finally make two more groups called CD4 Subsets and CD8 Subsets adding the third and fourth stain combinations respectively and set the color to green and the style to italic When you are finished creating groups click Close Click on the T Cell Analysis group your Workspace window should
50. changes The number to the right of each group tells you how many samples are included in each group Since there were four patient PBMC preparations each stained with each combination of antibodies each new group has four samples listed Click on each of the groups and verify that a different set of four samples is present in each of the different groups Finish by selecting the T cell analysis group 31 Remember that a group is a sample surrogate Thus any analysis that you apply to the group is applied to all of the samples in that group To see this in action click on the Lymphocytes gate in the lower Samples panel Hold down the ALT key while dragging with the left button so that the entire tree of analysis is copied and release the mouse when it is above the T cell analysis group in the upper part of the window Notice that several things have Eee happened Fir St the analysis File Edit Workspace Groups Windows Help Debug tree has been added to a row indented beneath the group name as if it were a sample 7 Fa pe Groups and Analyses Number q amples Second the analyses have been T CD4 Subsets tii CDE Subsets added to every sample in the T Experiment 1 i T PBMC Subset sample panel Third all Of v t prcenanaysis these analyses now appear in eae teal and bold which 1S the KE i Statistic Cells CySPE reagent Fluor reagent PhyEry reagent same color and style that you vi 931115 B02 S
51. cs text grids and backgrounds and puts them into a single window From here you can print a report publish to the web or generate a slide presentation Most importantly your layout is now ready for the next experiment all you have to do is add the samples to the Workspace and run the Batch processor the report will be generated without having to re generate the layout template And you can have as many of these templates as you wish for any Workspace 86 FlowJo Layouts Adv WspSAVE Wsp File Edit Layout View Align Help xjojsjejajajiajso Final Report Batch Complex Report PBMC Analysis Scaler Snares luntitled 1 This ends the tutorial There is more documentation available in the reference vveb pages which you ll reach from any of the Help buttons within the program by typing F1 anywhere in FlowJo or by looking at http www flowjo com v76 en windowstoc html If you have any questions or ideas for improvements please contact us at flowjo treestar com FlowJo Tutorial and Web Site are Copyright Tree Star Inc 1997 2010 Revision Date February 19 2010 Version 7 6 87
52. ctly the format you desire To demonstrate how fast this whole process can be there is a second experiment s data also supplied with the Tutorial in the folder Experiment 2 This is data for 14 more individuals and is therefore much more extensive than Experiment 1 Drag this folder onto the VVorkspace like you did for Experiment 1 and create the batch outputs as before With a well designed Workspace you can completely analyze and generate custom graphics and tabular reports in a matter of just minutes for an entire experiment Flowlo Layouts Demo Workspace wspt File Edit Layout View Align Help Debug SO Sj aa A ay QJ 0s scatter Gates Batch 1 ad uj abutting graph Full Report Header Footer Overlay example Scatter Gates IScatter Gabes Batch 1 This is the true power of FlowJo Experiment Based Analysis 10 Lesson 1 VVorkspaces and Basic Data Display FlowJo provides an integrated environment the Workspace for the viewing and analysis of flow cytometric data The Workspace contains a list of all of the samples that you load into it the gates that you apply the statistics that you calculate and the table templates and graphical layout templates that you design Think of the Workspace as your experimental notebook In general you will create a different Workspace for each kind of experiment that you perform A Workspace may contain multiple samples collected on various days a
53. ctly to the printer You can choose how many sets of graphs tiles you want on each page The forth tab will generate a PDF file with each tile represented on a new page The fifth choice is Write Web Page FlowJo creates a folder containing each tile as a separate graphic and creates an index html file to easily view the graphics from the World Wide Web The last tab will create a Quicktime Movie where each set of graphs becomes one frame of an animation You can play the movie in any movie viewer and cycle through all of the graphics All of the different views generated in these batch reports support printing saving to disk copying to the clipboard and creating Web pages a 8 nia i LI owo Layouts Tutorial Ws Chapter EJ vep Bile Edt Layout Objet Wes Ay Help Sjojsjejajaj ses re Liebe Batre 66 Now return to the new Layout view There is a Zoom control menu at the top of the window that adjusts the magnification of the view You can scale to any percentage such that the entire batch layout can be seen on the screen see above You will notice that FlowJo draws gray dotted lines in the window these correspond to page breaks in a printed document Select a small magnification like 12 5 to see many pages at once Note that as you change the viewing magnification the relative scaling of the graphs to the page boundaries do NOT change you are not changing the print mag
54. d change the axes to CD45RA vs L Selectin CD62L You will see four populations at least Create a rectangular gate around these populations as shown in the figure left The CD45RA LSelectin cells are naive the rest are memory cells You can name them M1 M2 and M3 Gl Lymphocytes CD4 931115 B07 Sample EN T X File Edit Graph Display Go Help fs godas AS Your graph should now show these four gates Close the Graph windows Your Workspace will show the subsets you just created including the differentiation subsets of CD4 cells which are a subset of lymphocytes which are a subset of the entire sample see below PhyEry CDASRA 4 20090303 1 mea Hialeah File Edit Workspace Groups Tools Windows Help Debug TIJ TEA k Options k Active Gate k Statistics Count 1561 10000 15 61 Groups and Analyses Number of Samples T All Samples 16 w 1 CD4 Subsets 4 Note that the CD4 gate and the CD4 subset ee j gates are not in green This is because these 7 Experiment 4 E gates have not been applied to the group TT PBMC Subsets 4 and therefore are not identical to a group vi di Cell Analysis 4 analysis They are drawn in plain black to e u ff LL denote the fact that these analyses are Name amp f Statistic Cels unique versions belonging to the sample a ae n mere The sample is in the group but the gates v GD CDA 15 6t 661 have not yet been applied to the group 60 Mi 2 79
55. d gate is the one with the square black handles drawn at each vertex The frequency of events in the currently selected gate within the entire sample is shown at the bottom of the window when you click on the Down arrow or double click on the gate the Graph dl window for the subset will be shown Click on the Down arrow to view the monocyte subpopulation Change the resulting graph to look at CD16 vs CD14 F Options k Active Gate Monocytes Monocytes22 00 k Statistics Count 10000 10000 100 096 25 Create two gates for the major populations that you see This time use the rectangular gate tool to create the gates Rectangular gates will be computed much more quickly but this is only evident when you work with very large files for instance more than 100 000 events You may find rectangular gates easier to create Create two rectangular gates like the graph below File Edit Graph Display Go Help Z Moraga OBS Name the upper gate CD16 CD14 dim ProMono since this may well be granulocytes you could name it Granulocytes if you wish The lower right population is principally mature monocytes name it Mono Look at the Workspace window You will see the new subsets displayed click on the ProMono subset and add the Freq of Parent statistic then do the same for the Mono subset The Workspace now looks like the Workspace shown below k Active Gate k Statistics Co
56. dit Layout Object View Align Help k ojxjejajaj Q 100 r 5 T e Overlay If you right click on the legend you will note that the popup menu now has an additional set r of menu items Overlay Scatter Analysis Unit Area FhyEry CD16 These are controls that let you set the histogram line and fill style for each subset If you right click on the Lymphocytes name in the legend you will see a popup menu as shown in the graphic on the image below T FlowJo Layouts Tutorial WS Chapter 7 wsp Ee Here you can select the line File Edit Layout Object View Align Help weights Hairline Normal Heavy xjojsjejajaj a 100 yaj ee ULJE or Thick the line styles Solid eats Set Control On si Dotted Dashed Long Dashed or oo Bee Complex or if the histogram Scatter Analysis Line Style Sopulation Name Line Weight Tinted should be filled None Filled or p Lymphocyt i E Sonoeves Tinted Select Filled Then right click on the line next to the green Monocytes color box and select Thick This will edit the line weight and the fill color of the two lines in the graph Unit Area Phyery CD16 After dragging the Monocytes line to the first place of the legend your layout should show the overlay histogram as shown below ini FlowJo Layouts Tutorial WS Chapter 7 wsp File Edit Layout Object View Align Help Kjojnjajajajqjuis xe women Overlay off Scatter Analysis
57. e overall output the page break and Select All Ctrl A4 Invert Selection Ctrl Slash 2175 Preferences Ctrl Save State to Preferences Ctrl Shift Y nl WS zi 101 150 CI geometry specification This layout Far specification can also be saved using the ese sammens U ngeted Save button on the menu itself too 67 In the Layout Window select the original Layout PBMC Analysis that has the two graphics as shown below These two graphics are a forward vs side scatter plot and a FITC CD14 vs Cy5PE CD45 plot In the Batch Report screen change the group to Experiment 1 Select to Iterate by Sample in the Iteration Type menu to enable the Batch button Click on the Batch creation button and specify that you want to make a new layout still four across The new layout will be added to the list on the left hand side Its name is the original layout followed by the suffix Batch It should look something like that shown below called Untitled 1 Batch E FlowJo Layouts Tutorial WS Chapter 8 vysp File Edit Layout Object View Align Help SENSOR BE tisl l PMBC Subsets Batch 1 PBMC Subsets PBMC Subsets Batch 1 Scatter Analysis Scatter Analysis Batch 15 FlowJo Layouts Tutorial WS Chapter 8 vysp File Edit Layout Object View Align Help Fjolla aja IG ej BES tjzl l PMBC Subsets I Scatter Analysis Scatter Analysis Batch 931115 B01 Sa
58. e A now will look like the one to the right Note that Fe edt Gach Display Go Help pe most of the events are CD45 positive and CD14 Pal negative this is exactly ad we expect for AJ OY O QA Go Se Aa lymphocytes in PBMC The CD45 negative population is probably red blood cells that contaminated the cell preparation Move this window to the side and click on the Up arrow button to make the parent population the active window You should be able to see both graphs simultaneously Fluor CD14 CySPE CD45 F Options gt Active Gate Statistics Count 7100 10000 71 0 FlowJo uses dynamic recalculation of gates This means that whenever you adjust a gate FlowJo automatically updates all visible windows to reflect that change Move the mouse over the lymphocyte gate in the Graph window the cursor changes to a cross If you now click and drag you will move the gate Move the gate so that it is over the monocyte population as shown below Gl Ungated 931115 B01 Sample OT fes TAX File Edit Graph Display Go Help sjojdJojsj Q FlowJo instantly updates the graph of the subset you will be able to see these dynamic changes in the graph window The new gate includes events that are all CD45 positive and a large fraction are also CD14 positive i e monocytes You can continue to move the gate around and see exactly how the gating affects the subpopulations These changes also occur when
59. e now identical see right Choa wsp FlowJo File Edt Workspace Groups Tools Windows Help Debug T F z E S Groups and Analyses T PBMC Subsetss w CD Lymphocytes Do E CySPE cv E CySPE Median Freg of Grandparent u Fren nj Parent Home Fq o 531115 B501 Sample 01 te GD Lymphocytes w ED CO5 GyaPE CD45 CW T CSPE CO45 Median Statistic ecells o 10000 77 00 F100 67 80 35 27 174 00 63 80 89 86 22 00 10 85 2 Freg of Grandparent Freg of Parent Ed Monocytes ED Mono Freg of Parent AF v GS ProMonro 9 65 Freg of Parent 43 86 1 9391115 B02 Sample 01 ics P Lyrmpliocyles w ED T Cells w ED COAT Freq of Parem wT Go COaT 22 25 gt Freq of Parent 46 99 ED Double Negative T 3 40 Y Freq of Parem 19 841 o 931115 B07 Sample 01 Its Ka LTE T8 22 4r d5 14 50 30 56 sja Tr 7O 1551 27a 37i 1 65 r OU w u 991115 B08 Sample 01 fts v ED Lmohocyies v o CDAT ED MI 6 42 P 24 70 eS Mp 362 ED Ma 6 49 GS Naive Faj 931115 001 Sample O2fes w 52 Lymphocytes w ED CD45 T CysPE CD45 CW CySPE CO5 Median Fa Ge fo 7T 34 05 176 75 Gy ade he pr oa Fima bn fe DE ANIES Gere ea ire lin I LEN i An unter ol Sapien dtl Samples C e 0 dd Shnet 4 Y ii COG Subsets 4 F Gass Ph Cap Pia raagan Phy rangar 991115 808 Sample 01 fes 10000 CODE L seleciin CO45
60. echanism for copying entire analysis trees if you hold down the ALT key as you drag with the left mouse button FlowJo will take the subpopulation you clicked as well as all of its descendants You will see this occurring via the shaded box that FlowJo draws which denotes all of the analyses that you are copying Click on the Lymphocyte gate press the ALT key and drag it to the C02 row Even though the children of the first lymphocyte gate are hidden if you close the triangle they are still copied Your workspace will now reflect the fact that you copied 8 different analyses gates and statistics with one operation 20090303 1 FlowJo The 1 e a r e m O r e C O mp le X W a y S O f File Edit Workspace Groups Tools Windows Help Debug selecting which analyses to copy For TIA es Ce instance you can shift click several A Z Groups and Analyses Number of Samples analyses to drag multiple different EETA T gates simultaneously or you can use l Name Statistic Cells CySPE reagent Fluor reagent PhyEry reagent J the control key to select several j Tem n serra e Seer aa ae Lymphocytes FF 7822 subsets and or statistics to be C A TT TT d d u 931115 B08 Sample 01 fcs 10000 cD8 L selectin CD45RA Tagge U 931115 C01 Sample 02 fes 10000 CD45 CD14 CD16 Th k h w U 931 115 C02 Sample 02 fcs 10000 CD4 aj Bs p CD8 e workspace now appears as shown on TD Lymphocytes 7891 the right You can delete analyses by TED
61. edian 114 00 Y Freq of Grandparent 63 80 Y Freq of Parent 89 86 w Monocytes 22 00 zan w amp Mono 10 85 Freq of Parent 49 32 w 65 ProMono 9 65 965 Y Freq of Parent 43 86 w 931115 B02 Sample 01 fcs 10000 CD4 CD3 CD8 en Se u W 931115 BO7 Samia e01 fcts 10000 CD4 L selectin CD45RA File Edit Graph Display Go Help TEE J u 931115 B08 Sample 01 fcs 10000 CD8 L selectin CD45RA u 931115 C01 Sample 02 fcs 10000 CD45 CD14 CD16 AF Canmanla Histogram Double click on this Lymphocytes subpopulation to open a graph of the lymphocytes within the 931115 BO2 Sample 01 file Q Now change the axis labels to view a histogram of CD3 Create a gate on CD3 cells and name it T E cells At this point your Graph window should look E Ne ee ere like the example on the left k Statistics Count 7822 10000 78 229 Fluor CD3 20 Name dh statistic Cells CySPE reagent Fluor reagent PhyEry reagent 10000 CD45 CD14 CD16 ad u AAs ue Sample Ta 10000 CD4 CD3 L aalantbim Click on the Down arrow to open a graph of the T cell subset Change the Y axis to PhyEry CD8 change the X axis to Cy5PE CD4 Create three rectangular gates 1 CD4 single positives 2 CD8 single positives 3 CD4 negative CD8 negative double negative T cells as shown to the right In the Workspace window click on the triangle next to the 931115 B02 Sample 01 fcs line These triangles open and close the
62. eground to yellow the background to none and type in values of 40 in the two scaling boxes Next click on the annotate tab to uncheck Show Annotation a check mark means that the attribute is On a blank box means the attribute is Off the Fonts tab change the Foreground font to yellow Graph Definition 31115 B01 Sample 01 fes Specify Annotate Fonts X Axis Forse Y Axis OrthSe Graph Type Contour Contour Levels 10 ay Foreground Show Outliers Background N C Use Large Dots Controls Checked items remain locked during iteration Double click to change setting 931175 B01 Sample 01 f 5 931115 B01 Sample 01 fos Scale Horizontal C40 Po yericah 9 FlowJo Layouts Advanced Tutorial Chapter 10 wsp File Edt Layout Object View Align Help vjojnjaj AJAJ Aj 100 Final Report Complex Report J Overlay Pale Subsets Freq Scatter Analysis Statistics Finally you can create some text boxes to annotate the graphs In this example the new text boxes were created with a white text overlaying a dark blue background Tip create one text box format it exactly how you want You can then duplicate it using copy paste or right click Duplicate Double click on the text box to change its contents For the final panel we will create two different tables The first table will have just statistics the second table will mix statistics and graphs In the table below yo
63. en expanded to a bracketed lt gt command The command within the brackets tells FlowJo exactly how to get the desired statistic i e which subset and which statistic are desired DO NOT edit the text within the brackets or else FlowJo may not be able to fetch the statistic value you want However you are free to edit any of the text outside of the brackets In this example select all of the text in front of the first bracket and change it to Lymphocytes are and after the second bracket add the text of the total events The window should look like shown on the next page 71 Text Properties Freq of Parent Statistic DataSet 1 originalDataset 1 path Lymphocytes CD45 name Freq of Parent ancestor Parent S Insert Keyword lt none gt vi For Sample 931115 B01 Sample 01 fcs v Font Dialog YI Color Size 12 Justify left vj Style plain Fill Color Line Color i Resize box to fit text Line Weight Hairline Line Style Solid i Text Properties Lymphocytes are sStatistic DataSet 1 originalDataset 1 path Lymphocyte siCD45 name Freq of Parent ancestor Parent of total events Insert Keyword lt none gt v For Sample 931115 B01 Sample 01 fcs si Font Dialog bu Color Size 14 MI Justify left M0 Style plan Fill Color Line Color E Resize box to fit text Line Weight E v Line Style Solid v To change
64. er e g Sample 01 Just by adding the data files to this workspace FlowJo Il Demo Workspace wspt FlowJo Fle Edit Workspace Groups Windows Help Debug has already accomplished most of the analysis steps for J Tr E Fa this data Each file was examined to see what Soaps and Analyses O T All Samples reagent panel was used to T CD4 subsets stain the cells Depending on tit CDS subsets u Experiment 1 the panel FlowJo added the TT PMBC subsets data file to one or more gt dit Teejis subsets Groups Then the gates TE San ai statistics or other analyses oe a a specified by each group were Y Freq of Parent 69 75 automatically added to the ba ED ay ee meg oF Parent F sample Thus gates and Monocytes 24 01 2401 statistics specifically w 931115 B02 Sample 01 fes 10000 designed for each reagent T T S pa pot A ce CE i i panel were applied only to v DOT 1387 1387 the sample tubes for which Fluo CD3 Median 45 59 Freg of Parer Ag 34 they were designed You can ane _ pe scroll up and down the F Fluo CDI Median 32 34 sample list to see the variety of different gates and statistics that vvere applied to each sample This workspace also has several graphical layout reports saved with it FlowJo saves not only analyses gates and statistics with workspaces but also any tabular and graphical reports compensation matrices kinetics and
65. es Experiment 1 and the od Experiment 1 16 samples are now listed in the bottom portion of the TEE E cj window The numters to the I 931115 B01 Sample OI fes 10000 al right of each group show U 931115 B02 Sample 01 fes 10000 U 931115 B07 Sample 01 fes 10000 how ery data files are U 931115 B08 Sample 01 fes 10000 included in that group You U 931115 CO1 Sample 02 fes 10000 U 931115 C02 Sample 02fes 10000 ee ne the Workspace U 931115 CO7 Sample 02 fes 10000 window as with any PC U 931115 CO8 Sample 02fes 10000 window you can resize the U 931115 D01 Sample 03fes 10000 F i U 931115 D02 Sample 03 fes 10000 group and sample portions H 931115 D08 Sample 03 fes 10000 by clicking and dragging on U 931115 D07 Sample 03 fes 10000 m the dividing bar between them Each file is shown as a test tube icon followed by the title of the sample s file To the right is shown the number of events that were collected in that file You can add additional columns to the Workspace display that show the stains used for each sample or any other keyword value found in the FCS data file You may also sort the sample list on the basis of any of these values by double clicking the column name 13 Edit Column Names To add the reagent list to the workspace view click on and select the command Edit Columns FlowJo shows the window on the right Move the desired column headers from the list of available attributes on the left to the l
66. et ven Aign Heb Iteration Popup menu vyhich is just below the Batch button at UTNE AS Sim a the right top of the Layout H Sete sass Editor Whenever you create a perra new Layout the current Iteration state for that view is set to Off When Iteration is off all of the graphs shown in the Layout are identical to the ones you dragged and dropped into the View originally FlowJo doesn t care what the current group is Select to iterate by Sample in the Iteration Type popup menu to enable the Batch button and to show the current iteration value Forse 931115 B01 Sample 01 fes Ungated 1nnnn a SION 63 Because the current group is All Samples FlowJo shows you the 16 different possible Iteration Values one for each sample in the current group You can see all of these values in the Popup menu Select one of the values from this popup menu for example the 931115 B01 as shown on the previous page By selecting a specific Iteration Value you direct FlowJo to display the graphs in the layout view as they look for the sample you just selected Therefore the graphs you now see are those for the 931115 B01 sample Note that if this sample did not have any of the subsets displayed in the Layout then FlowJo would show an empty placeholder for the graph Note the pair of arrow controls at the right edge of the window right below the Batch button You can use these to increment or decrement the current iteration val
67. eters you will type the percentile into the numerical box For now click on Freq of Parent and then click on Add Also add the Freq of Grandparent and add the Median and CV for Cy5PE select the parameter from the Parameter popup menu If you click on Help a corresponding web page will be opened with your web browser that gives you complete information about the various statistics When you have finished adding statistics to this population click the Close button to close the window Once you close the Statistics window your Workspace window will have several new entries as shown on the next page 22 Close The new lines begin with a statistics icon Click in the ileal File Edit Workspace Groups Windows Help Debug column name bar on the _ E e A i i vertical dividers Drag to show I5 AUH 2 6 the entire column names The Groups and Analyses Number of Samples en i All Samples 16 statistics icon is followed by the EN s parameter on which the statistic z pa s Name ah Statistic Calls Cy5PE reagent Fluor reagent PhyEry reagent 15 computed if applicable the w 931115 B01 Sample 01 fes 10000 CD45 CD14 CD16 Ja name of the statistic and the lg v Lymphocytes 71 00 7100 5 Y amp CD454 63 80 6380 computed value The Median Cy5PE CD45 CV 3597 Cy5PE CD45 fluorescence of the E OSPE CO4 Median Tam 7 Freg of Grandparent 63 90 C
68. ey look as though they should all be moved up since CD8 cells tend to express higher levels of CD45RA than CD4 cells Click on each gate and move them so that they more accurately enclose the populations as shown left Leave this window open for now Go back to the Workspace and open the graph for the CD8 subpopulation for Sample 02 C08 It still has the gates copied from the CD4 stained sample File Edit Workspace Groups Tools Windows Help Debug DUBHA Groups and Analyses Number of Samples lt i All Samples 16 b 7 CD4 Subsets 4 iit CD8 Subsets 4 w amp lymphocytes Y D COAT eD MI ED M2 E MI eD Naive tut Experiment 1 16 TI PMRS Subsets ae d ha Name a Statistic Cells 7 931115 B08 Sample 01fes 10000 v amp Lym hocytes 78 44 7844 Y amp COAT 24 0 24 0 Naive 931115 C08 Sample 0 10000 Y amp Lymphocytes 77 52 7752 Y D COBT 38 14 3814 ED MI 12 03 1203 ED M2 3 84 364 eD M3 O73 9 5 eD Naive 11 39 1139 931115 D08 sample 03 15 10000 Y amp lymphocytes 72 04 7204 o OAT 770 89 nana 38 Hovvever these are the ones you vvant to apply to the group Once again this is easy to do shift click the four subpopulations Naive MI M2 and M3 under the first sample and drop them on the CD8 T gate in the group In the Graph window for the second sample you will see the gates move to the new positions in the Workspace window all gates are now drawn in pink since all ar
69. f events in the entire sample that fall into the subset 18 If you double click on the graph inside the Lymphocytes gate FlowJo opens a new graph showing the data contained only within the Lymphocytes gate Also there is a new entry in the Workspace window see example below aj 20090402 T FlowJo File Edit Workspace Groups Tools Windows Help 0 0 z E e Groups and Analyses Number of Samples i All Samples u v Experiment 1 16 16 Y 931115 B01 Sample 01 fes eD Lymphocytes 71 00 7100 u 931115 B02 Sample 01 fcs 10000 CD4 U 931115 B07 Sample 01 fcs 10000 DA U 931115 B08 Sample 01 fcs 10000 CDS U 931115 C01 Sample 02 fcs 10000 CD45 U 931115 C02 Sample 02 fcs 10000 DA U 931115 C07 Sample 02 fcs 10000 DA U 931115 C08 Sample 02 fcs 10000 CDS U 931115 D01 Sample 03 fcs 10000 CD45 U 931115 D02 Sample 03 fcs 10000 DA U 931115 D07 Sample 03 fcs 10000 DA U 931115 D08 Sample 03 fcs 10000 CDS U 931115 E01 Sample 04 fcs 10000 CD45 U 931115 E02 Sample 04fcs 10000 DA U 931115 E07 Sample 04 fcs 10000 CD4 U 931115 E08 Sample 04fes 10000 CDS Coa L selectin L selectin Cos ER L selectin L selectin NEJ CDa L selectin L selectin CDI14 Cia L selectin L selectin Statistic Cells CySPE reagent Fluor reagent PhyEry reagent tn E 0000 SH eager Ph t ngen Fr nge CDS CD4S5RA CD45RA CD16 COs CD45RA CD45RA CD16 COs CD45RA CD4S5RA CD16 CDS GDASRA CD45RA
70. he pseudocolor plot this type of plot low resolution pseudocolor may be the best for slide presentations In general the probability contour plots give the best representation of the data though they are lacking in that they do not show rare events To work around this FlowJo provides the option of displaying Outliers Outlier plots combine the density estimation information of the contours with the rare event information of a dot plot The density plots either grayscale or pseudocolor are superior to dot plots in that they provide density information i e the number of events within an area by using different colors or shades Grayscale plots may be useful for publication and pseudo color plots for slide presentations Shown on the next page are examples of some of the other types of plots supported by FlowJo When you close a Graph window FlowJo records the plot style and axes When you next open the graph for that subset you will see the graph as it was when you closed it This is in part how FlowJo saves the environment across analysis sessions 16 eee oe ee File Edit Graph Display Go Help jes Q aj jojajzi l LIngales Ungated Ungated Cy5PE CD45 Cy5SPE CD45 k Active Gate k Active Gate k Statistics Count 10000 10000 100 0 k Statistics Count 10000 10000 100 085 I File Edit Graph Display Go Help ZJ Graph Display Go Help Oj 9 a 2185 jo M BAG Ungated an Ungateci T E Lo t c
71. he last column likewise for the CD8 subset Fgh aay To change the graphs to histograms of CD3 EST ener select the two cells with graphs and double oe Set eos click Change the X Axis to Fluor CD3 and Po 1115 B02 Semple ae the graph type to Histogram At this point your al T Layout Editor should look something like that e shown in the next page v cost E Floor CO3 Median 2 Freq of Parent Double Negative T x Freq of Parent S i 931115 C02 Sample 02 fes Co Lymphocytes ED T cells wT DT Fluor CD3 Median E Frey of Parent wv t coat 2 Fluor CO3 Median Freq of Parent 10 Double Neqalive T 2 Freq of Parent e an 85 H FlowJo Layouts AdvWspSAVE wsp SEX File Edit Layout Object View Align Help oj SN 4 a ej I Untitled 2 PMBC Analysis I Untitled i lUntitled 2 Statistics Histogram 031115 B 63 244 61 82 87 11 C D4 Cells 45 35 100 00 31115 00 60 41 68 05 50 50 45 Ga e KG 73 12 25 93 65 64 3 50 COS Cells 32 30 AT 95 sis rr 60 68 30 27 Ar aa 20 00 Novy you are ready to generate the full report for all samples Make sure to set the Iteration Options to Cells for the iterator and tubes for the discriminator and remember to select the All Samples group first any batch layout operation is only performed on the current group Click on the Batch button FlowJo compiles all of the graphs statisti
72. he two frequency statistics under the CD45 subset Drag the highlighted rows and drop them into the right portion of the Table Editor window The Table Editor will now look like it does here Lymphocytes CD45 Freq o randparen ymphocytes CD45 Freq o aren Just as you can use the control select then drag action on analysis trees between samples you can also use alt select then drag for transferring an entire tree to the Table Editor Foo Tables Unnted sjejj Click on the Monocytes gate in the Tle Edt Output Heb first sample and while holding down the shift key select any other subsets or statistics you wish to include Then drag the selected items to the Column Coum I Population Statistic anes Time Definition window in the Table Editor You will note that all of the analyses are added in order to the table the Table Editor should now appear as shown left When you drag a subset a gate to the table FlowJo assumes that the statistic you want is the Frequency of parent of that subset Sample v i Lymphocytes Freq of Parent Freq of Parent ea a Freq oF Parent L 3 Lymphocytes CD45 Freq of Grandparent Freq of Grandparent 4 Lymphocytes CD45 Freg of Parent aj 5 fo Moncytes rent FParent KE ent Freq of Parent S LS eee 7 MonoeytesjProMono Freq of Pare Freq of Parent 0 45 Table Generation OPRAN jj To create the output table turn ite
73. hocyte gate the Workspace window will show the group s CD8 gate under each of the member samples as shown here to the right You will notice that the subsets of CD8 cells are similar to the subsets of CD4 cells We will copy the same gates you used for the CD4 cells onto the CD8 populations you will then fine tune them 70090303 1 FlowJo File Edit Workspace Groups Tools Windows Help Debug jU e Groups and Analyses Number of Samples T All Samples 16 ja b 0 CD4 Subsets w i CD8 Subsets 4 v amp Lymphocytes d CDBT ii Experiment 1 ii PBMC Subsets w i T Cell Analysis 4 Name ai tatistic Cells w i 9391115 B07 Sample 01 feces 10000 v amp Lymphocytes 77 70 7770 w ed CD4 15 67 1567 2 79 279 w 931115 Cor Sample 02 f s 10000 Y E Lymphocytes 78 74 7874 w ED CD 16 59 1659 e Mit 1 91 191 ED M2 3 67 367 ED M3 1 32 132 ED Naive 90r 907 w 931115 D07 Sample dajes 10000 v amp lymphocytes 66 89 6689 w ED CD 22 97 2297 PREF ATA ATA Again FlowJo makes sure that every window that is open will reflect any changes you make The Graph window appears as shown below Select the CD4 group again Now shift click on the Naive M1 M2 and M3 subpopulations dj GF C E e NA 20090303 1 FlowJo File Edit Workspace Groups Tools Windows Help Debug Groups and Analyses Number of Samples e5 MI 65 Naive wit CDA Subsets w G amp D Lymphocytes Ed CD8T i
74. ht The organization of the Workspace window is more fully described in later chapters For now e Ses ie realize that the upper half of the window consists of Sample Groups which specify analyses gates and statistics to be applied to appropriately stained samples and the lower half of the window is the list of all samples in the currently selected Group The All Samples group always includes every sample in the Workspace p p p p Because this is a template vvorkspace there are no samples in it Adding appropriately stained samples will produce the analysis that the template was built to perform Dee Workspace ven Fowo AE There are several ways to load data into a ER Nora ek 1s eee He Da workspace perhaps the easiest is to drag the O 0 xt a Folder Example 1 into the FlowJo window annie e click on Experiment 1 in Windows Explorer u 0 and without releasing the mouse drag it over the lower half of the Workspace window Name A Statistic Cells When you release the mouse button FlowJo examines all of the files in the folder you dragged and loads all FCS data files it finds FlowJo can read data files collected on cytometers from any manufacturer The workspace now reflects the new samples and analyses see next page In this experiment each sample was named by the date of collection 931115 a code related to the antibody panel B C D or E and a sample identifi
75. i i LLI 0 m i Histogram 3 10 Fluor CD14 7 k Active Gate k Statistics Count 10000 10000 100 0 b Active Gate kx Statistics Count 10000 10000 100 0 Smoothed pseudocolor plot Histogram plot 17 Lesson 2 Gating and Statistics In this lesson you will learn how to gate data to create subsets You will also learn how to calculate a variety of statistics from the data Subsets in FlowJo are exactly like subsets in biology they representing a portion of the entire collection with specified properties e g Lymphocytes are those cells with low forward and side scatter You will always be asked to name a subset the name you choose is important for organization within FlowJo Select a name that is meaningful to you as this will help you keep track of your analyses Creating a gate is simple Choose the polygon gate icon fifth from the left K then just click inside a graph the mouse will appear as a cross hair and move the mouse Each time you want to change directions click This is the same process you would use to draw a polygon in any drawing program If O C a Zo you hold down the shift key you get unazes horizontal vertical or diagonal lines you can use the delete key to remove a polygon while drawing it Either click on the first point or double click at any time to close the polygon and create the gate File Edit Graph Display Go Help Open the first file in the s
76. ing the Add Statistic button and then just click and drag the new row to the other subsets to save time i e you are copying analyses from one population to another Note that dragging a statistic from one population to another is a way of copying the mathematical operation See the example on the next page You could also select all three subsets shift click them and add the statistic to all at once from the Statistics window 27 E 20090303 1 FlowJo Fle Edit Workspace Groups Tools Windows Help Debug Lo tee Groups and Analyses Number of Samples T All Samples 16 Name Jl Statistic Cells CySPE reagent Fluor reagent PhyEry reagent ae P 931115 B01 Sample 01 fes 10000 CO4s CD14 CD16 kl w 931115 B02 Sample 01 fes 10000 GA CD3 CD w amp Lymphocytes 78 22 7822 w amp T Calls 47 45 4745 w DAT 14 50 1450 Y Freq of Parent 30 56 w DET 22 25 2225 E Freq of Parent 46 89 w 55 Double Negative T 9 40 940 Freq of Parent 19 81 u 931115 BOr7 Sample 0115 10000 CDA L selectin CD45RA U 931115 BO8 sample 01 fes 10000 CD L selectin CD45RA u 931115 CO1 Sample 02 fes 10 CD45 CD14 Che we A second patient sample stained with the same reagents is found a few rows down labeled 931115 C02 Sample 02 Again we would like to apply exactly the same gates and statistics performed on the first patient sample to the second We could drag each line one by one to the second sample However FlowJo provides a special m
77. ist of visible attributes on the right Click Add Columns to display the settings Click Done to accept the changes and the Workspace view will add the additional columns to the table All Column values Statistic Cells Annotation File Name File Path Compensation Matrix Transformation Settings Gating Parameters Gate Coordinates La Graph Spec Sample State Columns to Display Name Statistic Cells Cy5SPE reagent Fluor reagent PhyEry reagent gt Add Columns gt 8 Remove All Default CySPE reagent Fluor reagent PhyEry reagent cell C T kaeT Done Cancel E 20090402 T FlowJo File Edit Workspace Groups j AO 2 m S Groups and Analyses Number of Samples T All Samples 16 7 Experiment 1 16 Windows Help To view the data for any sample double click on one of the lines in the sample list When you double click on the U 931115 B01 U 931115 B02 U 931115 B07 U 931115 B08 U 931115 C01 U 931115 C02 U 931115 CO7 U 931115 C08 U 931115 DO1 U 931115 DO2 U 931115 D07 U 931115 DO8 U 931115 E01 U 931115 E02 U 931115 E07 U 931115 E08 Sample 01 fcs Sample 01 fcs Sample 01 fcs Sample 01 fcs Sample 02 fcs Sample 02 fcs Sample 02 fcs Sample 02 fcs Sample 03 fcs Sample 03 fcs Sample 03 fcs Sample 03 fcs Sample 04 fcs Sample 04 fcs Sample 04 fcs Sample 04 fcs 10000 10000 10000 10000 10000 10000 10000 10000 10000 10
78. istics bound to Fluor Median E Pren s Parent the CD8 subset into the third row of Freq of Total gt Double Negative T the Grid S Fluor Median VI YT eee as ro ee c E Statistic Cele ple O1 fes gt 10000 75 95 7595 If you wish you can double click on be ee 46909 E aw E each item individually make sure that ni Saj FEF only one Grid cell is selected in order Gr CDA T 100_00 Fluor CDS Median 45 35 to edit the text remove the Freq of Pareri 100 00 b i di ee ar fota 14 18 annotations from the Text to leave Fhror COS Neda 42 30 E Fren of Parent 47 95 only the statistic for each cell Freq of Total 72 55 Se Double NHeqgathese T 700s Fluor COS Median 490 E Freq of Parent 20 05 Freq of Total aA U a1119 0072 Sample O2 tes ED Lymphocytes iF 6 GS T Cells Ta SH C CDA T 28 72 Fluor COS Median di Si Freq of Parent 28 72 Frer of Toti 16 40 SS CDA T 100 00 E Fluor COS Mediam 54 90 A we Chapter 10 wsp FlowJo To add graphs to the Grid click on the Ble Edt Worspace Groups Tools Windows Hep Debug population of interest from the Workspace T 07 9 e and drag it into the correct cell of the Grid Or Groups and Analyses Number of Sampler l if you already have the graph in the Layout gt gn pr i f Editor you can drag it and drop it into the e ate Grid Select the CD4 subset and drop it into pt t
79. j CDS Cells 831115 6 77 27 30 27 47 83 Note that any item in a Grid can be moved out of the Grid or to another position just right click on the item so the cell is selected and copy paste it to the new location Likewise you can take any item from that Layout View and drop it onto any empty cell in the Grid to make it part of the Grid To add graphs and statistics to the Grid you will select them from the Workspace and drop them into the appropriate positions in the Grid First create the appropriate statistic In the Workspace select the T Cell Analysis Group To a CD4 gate within the first sample add the statistic Median Fluor CD3 Drag this statistic to the Group s CD4 and CD8 gates so that it is applied to all samples in the workspace Your Workspace window should appear as shown in the next page 84 JE Em gets goms Topit shue Hep Now click on the Median CD3 statistic Eoee Le Ee ed under the CD4 gate and drag it to the Groups and Analyses Number of Samples Expeerinbernt 1 16 Fi second column of the second rovy of G2 Lymphocyies p Lu PRE Subrat lt a your new Grid drop it when that cell p pr is highlighted FlowJo creates a new A sien text box with the statistic and adds it Freq of Parent Free of Total to the Grid Add the Freq of Parent 2 CD T E Freq of Parent statistic to the next column Repeat the oor two drags for the stat
80. k the line and choose Properties from the pop up menu you can change the style of the line NJ o N a 4 3 53 FlowJo shows you the Object Properties window as shown to the right Object Properties Line Weight Change the line to be dashed by selecting Dashed in the Line Style popup menu and select the line to have arrows at both ends by choosing Double headed in the Arrow Style popup menu If you wanted you could also change the Line Color for the line Click on Line Style Arrow Style Hairline e Dashed w Double headed the button marked OK The dialog box will go away Your Layout Editor should now show the dashed arrow and look something like the one shown here FlowJo Layouts AdvWspSAVE wsp File Edit Layout View Align Help Jojejajaj ajer Untitled I jl T To create a text object click on the Text tool and then click anywhere in the Layout View To create a text box with defined boundaries click and drag the boundaries you want to have FlowJo displays the Text Properties window into which you can type any text You can also drag statistics from the Workspace and add keyword values by clicking on the Insert Keyword menu These items are live and will change as you create batch layouts for multiple samples Type PBMC Analysis into the window You can also change the font and style of the text in the Text Properties window Change the text colo
81. lar analyses You will copy the gates you created for the CD4 cells to the CD8 cells However you will have to adjust them then adjust the entire set of samples simultaneously taking advantage of group analyses Finally you will finish all the group analyses for the entire sample experiment You will then see how easy it is to modify the lymphocyte gate used by every sample with a single drag and drop action Use your existing Workspace or to better stay in sync with this text open the workspace named Tutorial WS Chapter 5 Let s move on to the CD8 analyses taking advantage of what we have already accomplished Li Lymphocytes CD8 T 931115 B08 Sampi E X 1 Again drag a lymphocyte gate the Fle Edt Graph Display Go Help J single population only don t select the O O a Z Qo child gates from any of the samples in LymphocytesiCD8 1 the T Cell Analysis samples panel and drop it onto the CD8 Subset analysis group 2 Select the group and open the graph of the lymphocyte subpopulation from the first sample 3 Create a CD8 T gate on the CD8 histogram much as you did for CD4 cells 4 Then show the contour plot of CD45RA vs L Selectin shown at the left 5 Move this window to the side where you can still see it and click on the Workspace window to bring it to the front k Active Gate P Statistics Count 2470 10000 24 7 36 6 Drag the CD8 T gate you created onto the group s lymp
82. le 04 fes 54 68 1 77 501886 SIR 83319 A 38 4507 Mean 63 70 88196 595 88196 25 06 47 48 4408 Standard Deviation The column heads shovy you the name of the subset s parent gates ancestry the name of the subset the statistic and the parameter on vyhich the statistic is calculated Each row in the table corresponds to a sample in the current workspace You may resize the columns by clicking on a column divider in the table header and dragging left or right The Mean and Standard Deviation of the statistics are displayed at the bottom of each column When the summary statistics are computed the numbers in the cells are drawn in bold italic if they are greater than one standard deviation away from the mean and in red if they are over twice the standard deviation away from the mean This will quickly highlight outlying samples making it easy to use the table editor to identify samples that are significantly different from the others in the group You can change this formatting by clicking on the first icon in the table editor shown right PE 46 You can save the Table in different formats CSV Comma Separated Values Excel HTML HyperText Markup Language RFT Rich Text Format SQL Structured Query Language and different XML flavors eXtended Markup Language Select the Batch button again Table Generation Options These options determine the appearance of your report fe O amp File Format
83. lectin CD4ERA events fall in the u 931115 B08 Sample 01 fes 10000 CD8 L selectin CD45RA U 931115 C01 Sample 02 fes 10000 CD45 CD14 CD16 Lymphocyte gate 63 8 of 931115 C02 Sample 02 fes 10000 Cp4 CD3 CD8 the Lymphocytes are CD45 931115 CO7 Sample 02 cs 10000 CD4 L selectin CD45RA positive 931115 C08 Sample 02 fes 10000 cpa L selectin CD45RA 931115 DO01 Sample 03 fes 10000 CD45 CD14 CD16 931115 D02 Sample ULES 10000 CD4 CD3 CD 931115 D07 Sample ULES 10000 CD4 L selectin CD45RA Now we will add some statistics to one of K ds i its these subsets In the Workspace click on the CD45 row so that it is highlighted Choose a statistic and any applicable parameters below Press the Add button to apply them to your analysis and then click the middle button in the rrerjbutton bar the Sigma button This 4indicates that you want to calculate a statistic on the currently selected subset You will now see the Statistics window as shown to the right On the left side of the Statistics window is a list of the statistics available Some of these statistics require that you choose a parameter on which to calculate the statistic for example Mean Fluorescence Median Mean Parameter Geom Mean Robust CV CV stdDev wile Freg of Total Freg of Grandparent Freg of Count Mode C Show Channel Stats Percentile Freq of If you wish to compute a specific percentile of one of the param
84. look similar to the one below When you select a group in the list of groups its contents are displayed below in the samples list Create Group ANpearance Name PEMC Subsets Color Gloup Narn n Syla tald lal ng Sample Inclusion Criteria Di Live group U Synchronized Include samples that use the following staring Emid COIS CD45 tia COS CDA L j l t n OAA CO4 L selechn COHSRA CES Selart _ More Choices s Equals k LI Show all Keywords in menus With reference bo samples in another group 5 Only choose from samples in Group No spechied group s e Also include all Each group is nk i iia in the upper group panel in the color and style you have chosen I 20090303 T FlowJo Cc Edit Workspace Groups Tools Windows Help Debug JO JE ej Groups and Analyses Number of Samples 16 T All Samples T CD4 Subsets T CD8 Subsets E Experiment 1 T PBMC Subsets 7 T Cell Analysis Name w u 931115 B02 Sample 01 fcs 10000 CDA4 CD3 cD8 Statistic s cells _ CySPEreagent Fluor reagent PhyEry reagent E Lymphocytes i ee i Ss ee w 9 T Cells 47 45 4745 wv amp CD4T 14 50 1450 Freq of Parent 30 56 w 9 CDST 2 2225 Y Freq of Parent w amp Double Negative T 940 Freq of Parent U 931115 C02 Sample 02 fcs U 931115 DO2 Sample 03 fcs u 931115 E02 Sample 04 fcs Right click and select Group Properties if you vvant to make
85. ly annotated data Of course another good practice is to save your workspace files The workspace does not have to be saved in the same location or even same disk as the data files the workspace remembers where the data files are 29 Lesson 4 Groups and Batch Analyses In this lesson you will learn how to take advantage of sample groups Groups are the primary mechanism by which FlowJo allows you to perform batch analysis You may create as many groups as you wish each group can consist of any of the samples within the Workspace and samples may belong to more than one group In general doing something to a group will be equivalent to doing the same thing to every sample in that group the advantage is that you do it all at once Remember that the default All Samples group always contains all samples in the workspace Only add analyses to the All Samples group if you want them to be applied to all the samples in the Workspace In Lesson 3 you learned that by dragging multiple nodes or entire trees of nodes you can accomplish the first step in batch analysis the ability to replicate multiple analyses simply and with the guarantee that the copies are identical to the originals But that still leaves the next step how can these analyses be applied to a whole set of samples at once To accomplish this FlowJo allows you to create groups of samples A group which is simply a list of samples from the Workspace can then serve as a sam
86. mple 01 fos ForSe Ungated E E TE BR Mc eee o Analysis S gh PBMC st Analysis a Ka ta 68 Now each tile consists of two plots the arrow line and the text item Remember that FlowJo creates a new tile for every sample in the current group One last important piece of information the Layout Editor view is live in that whenever you change a gate or analysis that might affect the view the view is automatically updated to reflect the change However the Tiled Report Web Report and Movie are NOT live If you create one of these reports and then change a gate the report is NOT UPDATED You will have to regenerate the batch output to reflect in these media the change you have made 69 Lesson 9 Generating Complex Batch Analysis In this chapter you will build on what you learned in Chapter 8 so that you can generate graphical reports that include statistics as vvell as graphical reports that dravy graphs from multiple different tubes for each tile This lesson builds on the VVorkspace you have finished from Chapter 8 alternatively you can open the workspace named Tutorial WS Chapter 9 Open the Layout Editor and create a new Layout named Complex Report Drag into the Layout View the first ungated sample 931115 B01 The ato o et Layout View should appear as Ford W911 E Simple 11 fes shown left Ungated VELA Tutorial WS Chapter 9 wsp FlowJo Mima File Edit Workspace
87. n this experiment __ reaserjrooter Change the magnification to 75 as shown here This gives you a better view of the Forward vs Side scatter dot plot If you wanted you could now cycle through every plot by turning iteration on and choosing Sample from the drop down list and clicking successively on the eS Next or Previous sample _ arrows just below the Batch am button However you can use FlowJo s Batch analysis to automate this process Click on the Batch Button in the upper right OrthSc From here you can choose from a variety of report formats Next to Place the tiles in type a 4 in the text box Choose columns Click Create to view the same gates applied to each of the samples inthe Ww 9 3 DH amp A Workspace e El Create Batch Report These options determine the appearance of your report Place the tiles in Then try out the different Batch button options Direct to printer to print out the output PO re m e Power Point Data to save the output in PowerPoint slide format e Movie to play each successive sample in frames of a QuickTime animation e Web Report to view the output in a browser gn asamp or Iterate by Sample e PDF to generate a report In the PDF file 6 pasebypani format Position the tiles on separate pages In Chapters 8 and 9 you will learn how to arrange aie ee the graphics in the Tiles view to be printed in exa
88. nd can provide a rigorous way to organize your data analyses To begin analyzing data in FlowJo you will need to create a new Workspace and add data files into it Later you can simply double click on this Workspace to continue work For this tutorial you will create a new Workspace and load a set of data files from a single collection You will learn how to carry out basic analyses including batch analyses At the end of the tutorial you will load a second experiment with a similar set of samples collected on a different day You will use all of the batch analysis tools you created in the first set of analyses and quickly derive detailed statistical and graphical information in the forms of tables and printed graphical layouts for both sets of samples Workspaces are the basic environment that FlowJo uses to help you organize your data They don t actually contain the raw data Instead Workspaces point to the data that you load If you move the raw data files to another disk FlowJo will ask you to find the data the next time it needs that information Workspaces do store much of the information about samples as well as all of the analyses gates and statistics that you have previously computed Introduction to the Sample Data Used in this Tutorial There were two experiments each performed on separate days The experiments involved 3 color immunophenotyping of PBMC preparations from human adults Each preparation was split into 4 aliq
89. nel of your layout view You may have to adjust the magnification in order to see the full area of interest FlowJo now shows the empty Grid placed in the Layout Editor La FiovvJo Layouts AdvWspSAVE wsp File Edit Layout Object View Align Help Xo SN a 4 a a Untitled 1 PBNIG Analysis Untitled Untitled 1 GJE OFF v CD 8 gate U Freq ofPa Freq ofP Freq ofPa Freq of Pa 61 92 67 11 23 21 68 05 50 50 45 63 2 73 12 28 93 65 84 3 3 Double clicking on the grid will bring up the Grid Attributes window Select a light yellow Fill Color and a dark blue Pen Color and enter the dimensions of 3 rows and 4 columns Grid Attributes Learn More 83 Columns Roas Finally it is time to start annotating the Grid contents Use the Text tool to add the titles Population Median CD3 of CD3 and CD3 Histogram to the first four cells in the top row Add the titles CD4 cells and CD8 cells to the second and third cells of first column Set the text attributes to bold text Your Layout Editor may appear as shown below I FlowJo Layouts AdvWspSAVE wsp et File Edit Layout Object View Align Help kjojsjejajaj aje ne Untitled 1 PBMC Analysis i Untitled TEER Statistics Samples Freq of A Freg of P Freg of A Freq of H Population Median CO of CDS CO Histogram 831115 5 63 24 67 11 23 21 7 02 O31115 C 78 70 28 93 65 84 f
90. nification Changing the orientation landscape or portrait can be easily done using Print from the File menu and setting the paper type and orientation you want You can also specify that FlowJo automates e ep to be as wiae as the graphic display This is pee PR a ee i done by selecting Scale H3 7 s To Page in the File menu ma s oe So ee of the window see right ca i If you select Avoid Page E gt Breaks from the same SULGU amp menu FlowJo uses your paper type shape but aoe A a e places tiles on the page such that tiles don t cross page boundaries gay 4 Once you have arranged the tiles exactly the way you want you can print kt OT e ee a them you will know i l i exactly how they will ges A Zs T dd AD appear on the pages Pa eee me 2 i E FlowJo MENEE Tutorial WS Chapter 8 vysp File asia Layout Object View Align Help ja X Cut Ctrl x h aj It might be cumbersome T to have to redesign the ge printing layout every time you create the batch output E HE 99 Ctrl C Therefore FlowJo lets you save the current pet T ai pe Clear elete state of the Batch Output with the Isat Layout simply select Save State to Preferences from the Edit menu see right This saves the values of the Page Setup paper type and orientation and the setting of the Layout Specification popup menu i e how tiles are to be placed in th
91. ometimes adjacent graphs have aia fle Edt Layout ew Aion Helo exactly the same Y axis or X axis FlowJo lets you remove the axis HEIN 8 4 4 ars labels to align the objects to be lisi wes adjacent to each other easily creating compressed graphical presentations suitable for publication Double click on the rightmost graphic and under the Annotate options choose to hide the Y axis numbers and labels and the X axis numbers and labels MG FlowJo Payouts RaVWepeRVE WED E i T i Now you can Move the right File Edit Layout Object View Align Help Kjo SB A a ajs mic graphic in the Layout Editor Bf of q side by side sharing a common Y axis Of course in this example the Y axis is different for the two plots however FlowJo still lets you put them side by side The layout will now appear as shown at left FlowJo s Layout Editor provides a few simple drawing tools to elaborate your graphical reports These tools can be selected by clicking on one of the icons on the upper left edge of the window as shown below You can choose a Rectangle tool to draw filled or unfilled rectangles a Line tool to draw lines and arrows a Grid tool to create grid shaped containers a Text tool to create text objects or a Zoom tool zoom in on a specific graphic Click on the Line tool and draw a line by clicking and dragging underneath the two graphics If you now double click on the line or right clic
92. out Editor to create reports You will learn how to create live text objects that contain sample specific information and statistics how to put in a backdrop containing for example logos or specialized forms and how to manipulate Layout Grids specialized tabular elements that can contain text tables graphs or any other items This lesson builds on the Workspace you have finished from Chapter 9 alternatively you can open the workspace named Tutorial WS Chapter 10 Open the Layout Editor and create a new Layout named Final Report Select 66 in the scaling popup menu at the top of the window chose View menu from the top menu bar and select Show Page Breaks so that you can see the outlines of the page breaks E FlowJo Layouts Tutorial W ae Edit Layout Close krl First add a backdrop From the File Menu select Insert Picture Navigate to the Advanced Tutorial Folder gt PC Workspaces select Image Files from the popup menu and T TAE P Tae open the file Report Backdrop jpg E goj ItShirE S View i Print Ctrl P Scale To Page Ctrl shift 1 Lookin Advanced Tutorial Scale To Width Cbrl ShiFk W Scale To Height Chrl Shift H Avoid Page Breaks Ctrl k ji Report Backdrop ipa Files of type Image Files Ka FlowJo adds the image to the layout This will be the template upon which you will add other elements like statistics and graphics in order to create the finished repo
93. ple surrogate That is you can apply analyses to a group much as you can apply them to single samples A group may consist of any number of samples from the Workspace Any given sample can belong to as many groups as desired However there is one rule about groups and group analyses that is inviolate every sample in a group has every gate statistic or other analysis specified by that group as long as it is applicable to Flips ih x the sample This will become clearer in the next few steps of the tutorial This lesson builds on the Workspace you finished in Chapter 3 Or open our premade workspace named Tutorial WS Chapter 4 The first step is to create a group that contains a set of samples which will receive similar kinds of analyses In our case we will begin by analyzing the samples stained with the second combination of antibodies CD3 CD4 and CD8 In the Workspace oy click on the second button in the button bar Create New Sample Group FlowJo brings up the window shown on the right Using this window there are a variety of ways to make new groups The middle portion of the window lists every different combination of Appearance Mame be sample Inclusion Criteria Color Group Name Style bold Live group C Synchronized Include samples that use the Following staining CD14 CD16 CD45 CD3 CDS CD4 L selectin CD45R 4 CD4 L selectin CD45R 4 CDS Select Equals i More Choi
94. r and font size as shown at right Select the text Color to be red and the size to be 18 Note the fill color is applied to the whole area of the text box the Line color is applied to the box drawn around the text box which is drawn only if the Line Weight is set to something other than None Click on OK to confirm the changes Your Layout Editor should appear as the example on the next page 54 Insert keyword lt none gt s For Sample 931115 BO1 Sample O1 fcs 4 Font Serif Color E Size 18 Justify left Style plain E Line Style Solid D Line Weight Fill Color Line Color Resize box to Fit text oa Gee 9 Flow fo Layouts Tutorial WS Chapter 6 wsp Ble Edt Layout Object View Align Help PBMC Analysiz Tmp el s a a a a i iair iiis i impia iici dp d Layout Graphics can be easily exported saved or printed To copy a subset of the items in the layout select the ones you want and choose Copy now you can Paste into any drawing program You can export the entire contents of the Layout Editor window by selecting from the menu File gt Export to App In Edit gt Preferences gt File Formats you can choose between different formats in which to export the contents gif jpeg pdf png svg or emf ay Print E Page lof 1 F LS Letter ow E S n x tin Orientation Landscape 9 Portra amp Rows 1 Cohn I 2 Mangus Left in
95. ration on by clicking on the Batch button near the top right of the window FlowJo will display the window shown left where you can select the group from which to build your table as well as a keyword as alternative iteration source etc Select Groups PBMC subset and Iterate by Sample Click OK and FlowJo will display a new window in which the table will be ee mss B shown The finished table looks like that hown below Iterate by Sample shown below I Use Heat Maps Iterate by Panel At this point FlowJo has cycled through every sample in the current group there Iterate by Keyword are four and has calculated each of the nine different statistics you requested on the applicable subsets If a sample did not have the subset required then there would we om be a blank entry in the table And if the CREN subset did not e the requested statistic node present there would also be a blank entry E Table PMBC Subset Freq Fle Edit FlowJo Help Lymphocytes LymphocytesicD Lymphocytes C LymphocytesicD Monocytes MonotytesiMona Monacytes ProM Samples Freq of Parent Freq of ParentFreq of Freq of Parent Freq of Parent Freq of Parent Freq of Parent 931115 B01 Sample 01 fes 69 00 91 17 62 91 91 17 21 00 47 00 43 67 931115 CO1 SampleO2fes 77 57 96 96 75 21 96 96 1458 39 51 47 BG 931115 DO1 Sample 03 fcs 53 54 92 87 49 72 92810 3130 4 08 40 139 931115 E01 Samp
96. rt The layout window should appear as shown on the next page For your own reports you can generate whatever design you wish including specific areas for text graphics signatures etc Simply design the form in any graphics program and save it and open it as you did this one Pi B FlowJo Layouts Tutorial WS Chapter 10 vysp File Edit Layout View KJOJNJEJAJAJ A 66 Align Help Obed tizl Untitled 1 Complex Reprot A Complex Reprot Batch 1 Overlay F MC Subsets PBMC Subsets Batch 1 Scatter Analysis Scatter Analysis Batch Untitled Untitled 1 Statistics In this report you will include three separate elements In the top panel you will create a text box containing information regarding the sample In the second panel you will put graphs that show the gating scheme used finally the bottom panel will contain statistics and graphs Because this layout will draw on information from multiple tubes for the same individual you will need to set the Iteration Options to Cells for the iterator and tubes for the discriminator If you don t remember how see the second half of Chapter 9 Begin the first panel by clicking on the text box tool the A button at the top of the window then click and drag a rectangular area that will fit to the right of the Sample Information text and within the gray area FlowJo brings up the Text Properties window Here you will select several keyword
97. ry time Use the Save button to set these choices as the default Select the FlowJo tab set the tiles to 4 columns set the Group to Experiment 1 select Iterate by Sample and click on Create 65 el Create Batch Report These options determine the appearance of your report beja eca rr Rows Place the tiles in 1 k Columns Order tiles Across ye Position the tiles on separate pages Group Experiment 1 Iterate by Sample C terate by Panel Iterate by Keyword ee see Las When you generate a Batch Output FlowJo will start with the first iteration value and generate a page layout or tile for that particular sample FlowJo then continues to generate a new tile for each successive Iteration Value until it has exhausted the current group These tiles are used to create a New Layout window such as that shown on the below Batch Layouts can be generated in six different modes as shown as tabs The first type will simply create a new layout in the layout editor with all of the iterated samples shown This is the most flexible way to generate a report as you can now edit the layout further adding titles or annotations to specific graphs or by removing graphs that are not interesting The PowerPoint Data option generates a PowerPoint presentation in which each tile corresponds to a slide You can ungroup these graphs in PowerPoint and edit them further The third tab directs the output dire
98. s that contain sample specific information for entering into the text box To insert a keyword value simply select it from the popup menu as shown Begin by selecting the keywords DATE date of sample collection CYT cytometer used for collection SYS the system on which data was acquired and Cells the sample identification In your own datasets you may have other keywords with important information that you can add Each keyword command is bracketed lt gt Do not edit any text within these brackets However you can add text between sets of brackets As shown in the next image you can type in explanatory text and hit return to create line breaks to format the text box Set the Fill Color background color to gray the text color called simply Color to blue and the Style to bold and Justify to centered 78 Text Properties key datasete 1 originalDataset 1 name DATE gt Key datasete 1 ori ginalDataset 1 name CyYT gt gt skey datasete 1 originalDataset 1 nam e SYS gt gt lt Key dataset 1 originalDataset 1 name Cells Insert Keyword tell For Sample EXP Font Serif Color Size 12 m Justify left jst Style plain Line Weight Hairline Fill Color Line Color Resize box to Fit text Line Style Solid Properties gj Note that the Fill Color applies to the line drawn around the text box should you Using Key
99. sh between samples matching in all of three criteria by determining a discriminator keyword in the iteration options In our example this would be the tube keyword which is different for each staining used PBMC T cells CD4 cells and CD8 cells thus allowing us to unambiguously identify a sample by the Cells and tube keywords Select tube as the discriminator as shown above Now you can select the different patients and have the graphs and statistics update in the layout view Set the iteration value back to Off This specifies that FlowJo will only show you the graphs and statistics from the originally dragged subset itt imt si The graphs and statistics i shown in the Layout View were derived from the first meas tact antibody combination Scatter Analysis k CD14 CD16 CD45 Now vated pi I mv we will drag in a graph ae ZS from a different staining combination Select the T cells subset from the sample 931115 B02 and drag it into the Layout Editor next LAMA to the first graph Your pi S layout should appear as ON depicted to the left KJOJSJBJAJGJ ajio n saj Complex Report ON of total events 931115 BO01 Sample 01 fcs Ungated 10000 Sample 01 fes 74 Add a text box item above the second graph typing in T Cell Analysis Select the text and setting the font size to 36 points Set line color to none and click OK The Layout View will look something like the next image FlowJo
100. sis Scatter Analysis Batch Untitled However first drag in three other graphs into the Layout Editor as shown on the next page select the T Cells graph from the second sample and the two Lymphocytes gates from the first sample within the CD4 amp CD8 groups in the Workspace Drag them all onto the Layout Editor one by one Your view will probably look something as shown on the next page 19 TO FlowJo Layouts Advanced Tutorial Chapter 10 wsp See You will now change the attributes on File Edit Layout Object View Align Help these four graphs Double click each pe ON BY A JAY J 75 GJEJ E E graph in turn tisi Final Report Complex Report In CySPE CQ4 931115 808 Sample 01 fos Lymphocytes 931115 B07 Sample 01 fos 7822 Lymphocytes 7730 When you are done click on the OK button All four items are changed as you requested Now move the graphs into different locations perhaps as shown in the example on the next page the lymphocyte and monocyte gates are shown first then the CD4 and CD8 gates on the CD3 subset and on the right the CD4 and CD8 gates Note that each graph is derived from a different tube The graphs may appear very dim at low magnifications however they will print just fine and if you scale up to 200 or greater you can see that they are drawn fine at high resolution 80 r As shown in the window below change several attributes First change the for
101. t that does not exist in the current Iteration sample then the statistic value is shown as n a Likewise a graph for such a subset would be shown as a Placeholder The final topic in this Chapter deals with Layouts that derive graphs or statistics from different samples tubes This will only be useful if your data files are properly annotated As you learned in Chapter 8 FlowJo forces all graphs to be derived from the same sample during iteration To create a batch output FlowJo forces the iteration value to cycle through all possible values for the current Group By default this means one Frame for every sample tube in the Group However you may want to generate graphic reports wherein each Frame derives graphics from multiple samples For example you may want to generate one frame for each patient and therefore iterate by patient ID Alternatively you may want to iterate such that each iteration value corresponds to a different tissue studied from an animal where you have performed multiple stains on the tissue samples In these cases you would like to Iterate not over samples or tubes but to iterate over Patients or Tissues If your FCS data has keywords that contain such information then FlowJo gives you this ability If you don t know how to add this information to your data ask your local Flow Cytometer operator or Instrument Sales Representative For the demonstration in this tutorial the data supplied has a Ke
102. the particulars of the text display select a red text color and 14 point font size You can click on the OK button to accept changes and go on Note that you can also select keyword values to add to text boxes the keyword values are data encoded in the text box file itself You can create additional text boxes with more statistics by dragging them from the workspace You can also add multiple statistics to one text box by selecting multiple statistics and dragging them into the Layout View Again you are free to edit the text outside of the brackets don t edit text within the brackets Now click on the text box and drag it so it overlays the graphic item in the Layout View The view should look something like what is shown below E FlowJo Layouts Tutorial WS Chapter 9 vysp File Edit Layouk Object View Align Help Kjojsjejajaj jos mimi Complex Reprot Overlay PBMC Subsets PBMC Subsets Batch 1 I Scatter Analysis Scatter Analysis Batch Complex Reprot Lymphocytes are 69 22 of total events E 931115 B01 Sample 01 15 Ungateci 10000 Zz Ha Note that like the graphs statistics in the Layout editor are also live If gates change causing the number of events to change then they are automatically updated 72 In addition Statistics respond to the Iteration value if you select a specific sample to view then statistics are shown for that sample If you select a statistic of a subse
103. through different samples in the Workspace and how to create a combined output for printing or export of every graph for every sample This lesson builds on the Workspace you have finished from Chapter 7 alternatively you can open the workspace named Tutorial WS Chapter 8 and drag in the Experiment 1 data Open the Layout Editor and select the Scatter Analysis Layout When you drag items into the Layout View FlowJo by default shows you the desired graph for the sample from which you dragged the subset However FlowJo can show you the corresponding graph from any sample in the current group To do this you must select an Iteration Value corresponding to the desired sample First what is an Iteration Value In order to perform batch processing FlowJo cycles over every sample in the current group i e whichever group is selected and displayed in the workspace Thus in the All Samples Group for this experiment there are 16 samples and there are 16 different iteration values for the group one for each sample In the CD4 Analysis group with four samples there are only four iteration values corresponding to the four samples in that group Normally there is a one to one correspondence between an iteration value and a sample in the group In Chapter 9 you will learn how to iterate by other criteria to create more complex reports The Current iteration value iS prema e rena ER displayed and selected in the ffe tdt tavo Obj
104. ti color dot plot shown left Scatter Analysis a a oO 5 u CySPE CD45 Di FlowJo draws two dot plots one for each subset in the same graph In addition it automatically creates a m SEE E Legend for the overlay shown to the QU ee right of the graphic You can change the Legend properties by double clicking Sen on the legend Add the Population Name by choosing it from the Insert popup menu and remove the Sample Name by clicking on it and pressing the ritcoo tnewt Harin delete key If you click on Save this settings will be applied to your preferences Click OK vi Style plan v You can change the color of any subset by clicking on the color box next to that subset in the Legend text box You can change the stacking order of the colored layers by clicking on any item and dragging it up or down in the list Finally you can delete an item from the overlay by clicking with the right mouse key on the subset and choosing Remove layer You can overlay an almost unlimited number of different subsets on the same graph Note you can choose to show the Legend box for any graphic even if it is not an overlay from the Annotate preferences in the Graph Definition Window There you can also set color and line styles for single graphs just as you can for overlay graphs As noted before all Layout graphs are live This means that if you change a parent gate for one of the subsets the graph fe
105. titled Forse 931115 BO1 Sample 01 fcs Lingated 10000 simultaneously view many different subsets or even multiple views of the same subset while moving a gate used to define that subset Lo FlowJo Layouts AdvWspSAVE wsp File Edit Layout Object View Align Help xjojsjejajaj ajr Meo v 831115 B01 Sample 01 fes Ungated 10000 931115 501 Sample 01 fes ngate 10000 i To create another view of the same subset you have four choices 1 drag the same subset from the VVorkspace vvindovy into the Layout Editor again 2 select the first graph in the Layout Editor and do a Copy and Paste operation 3 right click and select Duplicate or 4 Hold the Alt key while clicking the graph For now duplicate first graph and move it over to the side You will note that the second graphic is identical to the first 51 Graph Definition 131115 B01 Sample 01 fes Specify Annotate Fonts X Axis Fluor CD14 Y Axis CySPE CD45 Graph Type Pseudocolor Contour Levels Foreground C Show Outliers Background Controls Checked items remain locked during iteration Double click to change setting 931115 B01 Sample 01 f es 931115 B0O1 Sample 01 fes Scale Horizontal 100 Vertical 100 To change how it looks double click on the graphic FlowJo shows you the Graph Definition dialog as shown left From this window you can specify exactly how you
106. u see in the Final Layout was generated by adding a FlowJo table Open the Table Editor and select your previously created T Cell Subsets table Batch to create the table and add to Layout as you did in Lesson 6 You should see the table in the Layout Editor MEI FlowJo Layouts Advanced Tutorial Chapter 10 wsp I File Edt Layout Object View Align Help PR NS Ba ajaj AJ 100 Final Report Complex Report Final Report Statistics 8 1 SI ES e Freq ofParei Freq of Pare I FlowJo Tables T Cell Subsets File Edit Output Help Next resize the table to make it E AL ag ean J smaller Click on the lower right 1 sore M hand selection handle and drag it at Ap Column mopman i zae Parameter Name PRR upsets 1 Lymphocytes T Cells Freq of Parent Freq of Parent to make the table fit in the area of _ revessses Frea E 2 LymphocytesiT Cells Freq of Parent Freq of Parent 1 the Layout Editor You can not resize individual rows and columns by clicking on any Lymphocytes T Cells Freq of Parent Freq of Parent 4 dividing line and dragging Lymphocytes T Cells Freq of Parent Freq of Parent 5 I Lymphocytes Freq of Parent I Freq of Parent 2 I Lymphocytes T Cells Freq of Parent 1 Freq of Parent 3 ILymphocytesjT Cells og of Parent I I Freq of Parent 6 ILymphocytesjT Cells Freq of Parent Freq of Parent 7
107. ue by one i e you can use them to cycle through successive samples in the current eroup Switch back to the Workspace mye Tutorial WS Chapter 7 wsp FlowJo and select the CD 4 Su b sets File Edit Workspace Groups Tools Windows Help group see right In this group T AE E there are only four samples EEDE m roups an nalyses umber of samples T All Samples 16 b i CD8 Subsets wil ut Experiment 1 Lymphocytes b tur PBMC Subsets b 7 T Cell Analysis Name Statistic Cells w u 931115 B07 Sample 01 fcs 10000 w amp Lymphocytes 78 37 7837 w amp CD4 21 18 1660 ED MI 19 22 319 CD M2 461 E MI 9 154 Naive 9 6 653 w j 931115 C07 Sample 02 fcs w amp Lymphocytes Ie FlowJo Layouts Tutorial WS Chapter 8 Wsp j Now if you select the iteration popup Se ie ee ee Be eee menu from the Layout Editor window xjojnjejajaj ajo vaj Gj j jaaj aji you will only be given a selection of some v ss1nis 07sampeorts ve four different samples to choose from riers yey see left Again this is because HOSE egies whenever iteration is not off FlowJo looks through the current group to decide what possible samples can be displayed 100 ForSc 64 La FlowJo Layouts Tutorial WS Chapter 7 wsp File Edit Layout View Align Help Note that if you construct a Layout Kjojsjejajaj aje ne oMa for a specific group which has unique tjsj Scatter Analysis lOverlav I
108. umbers are machine specific so see Ropistor License e you need a serial number for every we ee PC on which you will run the program We provide free PuchaseLicense ou de net own the erosram tut evaluation serial numbers that let you run the program for a specific Otherwise you ll probably want to time period generally thirty days request a free 30 day evaluation license In this way you can try out FlowJo ha your own data files if you have been issued a serial number enter it here and When you first run FlowJo you will click the button on the right to activate it be presented with the dialog window shown on this page Once you enter a serial number FlowJo won t ask for it again For now ee you can click the Run In Demo Ggngonyiheprovdeddetafics Lau RuminDemoMode Mode button and continue Later you ll want to request your Hardware Address 001427E6B836 Quit own serial number to try out the program with your own data files For more information about registering point your web browser to www flowjo com try html or send an email to FlowJo treestar com SA must be 16 characters 15 Demo Workspace wspt FlowJo File Edit Workspace Groups Windows Help Debug B Groups and Analyses Number of Samples a E All Samples i CO4 subsets ur CDS subsets i PMBC subsets ut T cells subsets Open the Demo Workspace by double clicking on PC Workspace wspt in see the window at rig
109. unt 2200 10000 Li 20090303 1 FlowJo File Edit Workspace Groups Windows Help Debug T OF Da e LO Note that the Monocyte Groups and Analyses Number af Samples is subset is indented one All Samples 16 Df mm level as is the Lymphocyte Name i Statistic Cells CySPE reagent Fluor reagent PhyEry reagent subset Both populations v U 931115 B01 Sample 01 fes 10000 CD45 CD14 CD16 are subsets of the sample amp Lymphocytes 71 00 7100 a w ED DAS 63 80 6380 The Mono and ProMono FE ED Cy DE Y Cy5PE CD45 Median 114 00 subsets are indented to a Y Freq of Grandparent 63 80 second level and are Y Freq of Parent 59 86 v Monocytes 22 00 _ shown below Monocytes w amp Mono 10 85 SES EBA TEF because they are subsets of 7 ProMono 9 85 monocytes This hierarchy Y Freq of Parent 43 86 g a U 931115 B02 Sample 01 fes cD3 CDE of this gating 1s visible u 931115 B07 Sample 01 fcs L selectin CDOD45RA E LI 931115 R08 Samnle i tes i i 4 L selertin L DARA bal from the VV O r k S p a C e window 24 This hierarchy is important in that it affects how FlowJo works It is analogous to a genealogical tree in that each subset is like a child of its parent population Thus in this example Lymphocytes is a child of the sample and a parent of CD45 and a sibling of Monocytes FlowJo does not allow you to name siblings i e gates on the same population with the same n
110. uots to be stained with four different combinations of antibodies see table thus there are a total of four tubes per PBMC preparation In general we refer to each tube or data collection as a sample In this experiment there are four samples for each cell preparation In this experiment there are four patients in the second folder Experiment 2 there are 14 The stain combinations used are as follows 11 As you work through this tutorial the goal is to analyze the frequencies of some of the subsets that can be identified using these antibody stains and to collate graphs and statistical information about subsets Once you have launched EAD z FlowJo you will be shown File Edit Workspace Groups Windows Help Debug 7 a window similar to that shown to the right Note Groups and Analyses Number of Samples 0 the Help Menu near the tFaisampies top right Most windows in FlowJo have a Help ee menu or a Help button When you click on Help se eee FlowJo launches your IJTag sam ple s Here standard web browser to get the on line version of Help or you can press the F1 key on your keyboard at any time The help for FlowJo is HTML based when you ask for help you will automatically be shown information about the active window Within the browser you can navigate to all of the help pages for FlowJo and learn how to operate the program You can go directly to the main FlowJo help page by selecting
111. you make minor modifications to a gate like clicking and dragging a single vertex 100 200 u Forse v TH k Options k Active Gate Lymphocytes Lymphocytes22 15 r Statistics Count 10000 10000 100 096 20 Move the gate back to the lymphocyte population so that the gate accurately reflects lymphocyte cells Change the graph for the lymphocyte population double click on the gate in the graph window to get to the Lymphocyte Graph or simply select the corresponding Graph window so that it displays a histogram of CD45 Select Histogram using the Y axis pop up or use the Options disclosure triangle to define the graph type 5 Lymphocytes 931115 B01 Sample 01 fc jem File Edit Graph Display Go Help JON S28 288 The graph should now appear as shown on the right If you wish you can change the Y axis scaling on univariate plots through the controls in the options opened by the options disclosure triangle Histogram Creating a gate on a histogram is the same as creating a gate on a bivariate plot click and drag in either direction Make a gate to include only the CD45 positive cells aes 937715 BOT Sample OT fe k Options File Edit Graph Display Go Help i Xie JAZZ I Lymphocytes Z b Active Gate H Statistics Count 7100 10000 71 0 Select the range gate tool E and click on one side of the peak and drag to the other side VYhen you let go of the mouse
112. yword Field Cells which has a unique value for each different Patient sample In the demonstration data there were four patient samples ID0001 to ID0004 that were stained with four different combinations of antibodies The Experiment 2 Folder has Patient ID0005 through ID0018 which you may wish to analyze later as a larger data set I FlowJo Layouts Tutorial WS Chapter 9 wsp We will now tell FlowJo that a it should iterate over patient Es E Mi samples FlowJo has a menu 8 where you can specify which Iterati FCS keyword should be used as the controller for Iteration Select Cells which for this dataset contains the unique patient ID Lymphocytes are 89 22 of total events Forsc E E 931115 B01 Sample 01 fcs Ungated 10000 E u 73 After you choose the iteration fae parameter FlowJo will search File Edit Layout Object View Align Help through every sample in the joj jajajajjajios Ya OS Fayence current Group and build a list jj center cels vy 100 sabe of unique values of the Emmm 2 Keyword Cells found in the pyes PBMC Subsats Batch 1 current group The current pac Scatter Analysis Batch workspace has four unique values of Cells which are shown by clicking on the iteration menu see image to the e h Lymphocytes are 89 22 ng t of total events 50 100 Forse a a 931115 B01 Sample 01 fcs Ungated 10000 It is further possible to distingui

FlowJo Advanced Tutorial - Flow Cytometry Core Facility

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