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FlowJo - CMIMA

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1. you have a Serial Sumber ay t Finin eer irent FlowJo 1S distributed over the lease eegerit in che bow hele and click on sa SE Internet in a state where it can only ey read demonstration data You can Hyoar cite bas ametwork dongle that providesuser 2 i licenses over the loca netwerk click on Wine Behari run this tutorial or look at special Dangle and Flaw ae ta Gere the ey Care FCS files that we provide but it will Pie lets tL oo 1 You may also npn Apaioa in Gemonstretion Meie ick not read your data until you have OF TER OS DEAE n wide go begin 2 eH g Fra pi obtained a serial number from Tree Star Serial numbers are machine specific so you need a serial num ber for every Macintosh on which you will run the program We provide free evaluation serial numbers that let you run the program for a specific time period generally sixty days When you first run FlowJo you will be presented with the dialog window shown on this page Once you enter a serial number FlowJo won t ask for it again Note The FlowJo application provided on the Demo Disk is already in Demo mode you won t see this window For now you can click the Demonstration Mode button and continue Eee ahe ducer ates Introduction 5 Later you ll want to request your own serial number to try out the program with your own data files For more information about register ing point your web browser to www flowjo com or send email t
2. nodes are analysis rows in the Workspace window s When you delete Confirm action the lymphocyte Deleting nodes also deletes all analyses belonging to these gate all of the subsets children Are you sure you want to delete the 2 selected node s subsets of lym phocytes will also be deleted E avay use same answer NO _ es_ this is because those subsets have no mean ing without a lymphocyte gate being 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 When you delete a gate FlowJo lets you know that all of its descendants will also be deleted in the confirmation request 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 30 Lesson 3 Copying Analyses to Other Samples drawn above were created on a sample that had PE CD8 and Cy5PE CD4 If you were to drag this population to a sample where you did not collect a Cy5PE channel then FlowJo could not copy the gate and noth ing would happen However if you copy to a sample that has both PE and Cy5PE but the stains are different then FlowJo displays a warning For example drag the CD4 T gate from sample C02 and drag it to sample C01 FlowJo presents you with anothe
3. fa Tutorial WS COharber WH Table Ed Hnr at rare is BB Feet cages EE Tc ress gh aa T ED lymp ETH eegerecy E toreghecytec ETA E Lipit En T Pii Freg ai Patki EE Lawet DET E Erepir E mpaya OR TA Majin E Large tee oie T Bregat Pieri E Lede TEE TI Frega G Usinge Teo heita E uyprgtecytea tbe Tas Freg t Peram Ey Titae CE T Tiie iregi E tyrrerenaies 0h T Miere medii i Lope LG T Miere Freg af Peruri Remember you can apply a table template to any group this could be useful for separately analyzing different experiments that you have grouped separately If you apply a table to a group that has no gates as requested by the table you will get a lot of empty values Tables can also be added to Layouts graphical layouts using the Layout Editor Simply drag a named table from the left portion of the Table Editor window and drop it in the Layout Editor 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 FlowJo creates tables in the background letting you perform other analyses
4. tic tells you what the frequency of CD45 events is within the parent subset Lymphocytes You will note that the value 89 8 is equal to 63 the frequency of this subset within the sample divided by 70 5 the frequency of lymphocytes within the sample The Freq of Grandparent shows you the frequency of this subset within the subset two levels up in this case the sample itself Thus for this subset ee the Freq of Grandparent is the Ai j art 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 Next you will create another gate on the sample this time for mono cytes Open the graph for the entire sample choosing the Forward vs Side scatter display option Draw a gate around the larger population as shown to the left and name this subset Monocytes Lesson 2 Gating and Statistics 23 Dem hamipie dirs seers SE Note that the currently selected gate is robs Biers DE VEER IETA e the one with the handles drawn at each Ejus vertex The frequency of events in the currently selected gate within the entire sample is shown in the upper right portion of the window when you click on the Down arrow or double click on the gate the Graph 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 CD
5. SS LICO Semple Oe tes A di FEN TS E Re Sample De Tos Ait SED TD prap 0E Tie SE TOOR Sanpete BEL PS DOT Gannple OF tos FEN S Di Gap TE Tes SEL BS E00 Sample 04 tos BEL 1 PSO Shatin le Oe tis FT RSE Shanna ie Oe eee eS BOR Sage OM tes 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 C02 as shown above By selecting a specific Iteration Value you direct FlowJo to display the graphs in the View as they look for the sample you just selected There fore the graphs you now see are those for the 931115 C02 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 Lesson 8 Creating Batch Graphical Reports 73 below the Help button You can use these to increment or decrement the current iteration value by one i e you can use them to cycle through successive samples in the current group Switch back to the SS itoni ws onapter E Workspace and select BEREG the CD4 Subsets group ir eee EE see right In this group is ptet there are only four samples Bip ee Cr T Now if you sele
6. 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 instan taneous feedback for gating operations where you can simultaneously view many different subsets or even multiple views of the same subset while moving a gate used to define that subset To create another view of the same subset you have three choices 1 drag the same subset from the Workspace window into the Layout Editor again 2 select the first graph in the Layout Editor and do a Copy and Paste operation or 3 select the first graph and hold down the option key while you drag it this creates a copy of the object and then lets you position it For now option drag the first graph and move it over to the side see next page 58 Lesson 7 Creating Simple Graphical Layouts You will note that EEA p peee t a Y as pata a E Cie i G fee the second graphicis ma OOO A identical to the first To change how it looks double click on the graphic FlowJo shows you the Layout Item Definition dialog as shown below From this window you can specify exactly how you want the graphic to appear As shown in the example change Laromt item Definira Barple Os ES Sa Od ea Papell its the X a
7. E E i we WD sane eee dreke tes a tuppheag tea BED Fee tn cii F Es freg of Pie fp cag rT E Dreg oot nA ED pait nepite T o Ermi m E arns Graphs 02 fen SE quanehooy ter Lesson 4 Groups and Batch Analyses now applied to all of the CD4 subset samples When you click on the CD4 sub set group you will see the four samples with the lymphocyte gate now drawn in green italic as shown on the right Double click on the first sample s lymphocyte subset and change the graph to show a histogram of CD4 Create a gate for the CD4 T cells like the one shown below Click on the Down arrow in the box at the upper right corner of the Graph Window in order to view the population defined by the active gate Now you are looking at the CD4 subset within the lymphocyte gate To show the graph on the next page without gates 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 on the next page Remem ber to create a rectangular gate hold down the option key as you draw the gate The CD45RA L Selectin cells are Naive the Tutorial W3 Chapter TE anata Sre Di res ice eng AAE oe eg nog Saree OFA Fe aT la me EO afer Saroe OE Fes ei wa Sua pe es rence 2 aae bareple Dlia iymphargins E 1 jeer 17 Mok TAE mar maj masar bep rest are memory cells You c
8. 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 EIH window by selecting one of the Action buttons shown left they are near the top right corner of the window The first button is an icon of an application This button will save a PICT file to your disk tell the Mac to launch another application as set in the Layout section of the Preferences and direct that application to open the file The second button is a disk if you click on this button the graphics are saved as a PICT file on your Macintosh any drawing program can open this file The third button causes the graphics to be copied to the Clipboard you can then paste them into any drawing program Finally you can choose the Printer icon to print the graphics directly to your printer If you choose to Print you may wish to change the Page Setup from the File menu or by clicking on the Setup 64 Lesson 7 Creating Simple Graphical Layouts button to orient the page and change the magnification as you desire The light gray lines drawn in the Layout View represent page boundaries for printing you can use these as a guide to set the Page Magnification appropriately You can also copy items from layout to layout within the same work space Click on the first graphic only and choose
9. Pra Paras Fire ueniry E Penieyten Prahan Frey of Parent When you drag a subset a gate to the table FlowJo assumes that the statistic you want is the Frequency within the sample of that subset To create the output table click on the table button near the top left of the window FlowJo will display a new window in which the table will be shown If you analyze a large workspace you may first see a progress monitor window as FlowJo builds the table see bottom right You can cancel the table build by clicking on Stop As FlowJo calcu lates the values for the table they are filled in During the calculation process FlowJo displays a Progress Bar near the top of the Table Window to show you how far along it has gone at any time The fin ished table looks like that shown on the next page At this point FlowJo has cycled gnn Flowde Tasks through every sample in the cur Tasks 1 Memory 10 Mb rent group there are four and Building Table EA AENEA 52 Lesson 6 Tables and Layouts Collating Data Output Tote ral WS On aptor O6 FE MC Subsets TH 1 21 Semple Ofer 2A117001 re fes 28i at Sap 02 tes ES 1 13 000 Same Ol Por Fiap Zid Drt 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 be a blank entry in the table And if the subset did not have the requested s
10. a control button FlowJo dis plays a description of that control s action esist ame m ier is unt ta the wurtapace in the button bar The second part of the es steiner Workspace above the central divider is a aaan list of the current groups Later you will see how to group sets of samples together for batch analysis The bottom part of the CEEE window includes a list of the samples in ee ET T the Workspace Bemire hdd reenter n Tararrear Paalma eee oe eee Now click on the left most button Add DataFiles to Workspace You will see a standard Macintosh Open File dialog navigate to the Sample FCS Data folder that you downloaded with the tutorial see above fa Untied Werkepmce I ARATA Highlight the folder Experi ment 1 and click on the button at the bottom to add all of the files in this folder 16 at samples one for each staining ERII THEI Dampi Ti fu EN SD Seog ALda fier tube four sets of PBMC PO INi AOS are tee SURO Derm tt fe LO with four stains each Your T a eaten 5 Workspace window should ami 15 Lor i T mna Eza te fen i j3 look as shown 12 Lesson 1 Workspaces and Basic Data Display Two things have happened FlowJo created a new sample group with the same name as the folder containing the FCS files Experiment 1 and the samples are now listed in the bottom portion of the window The numbers to the right of each group s
11. any empty cell FlowJo automatically creates a Text Box in that cell Use this feature 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 Select all of the cells in the Grid and change the text attributes to a 9 point font with Bold text Resize the columns and rows to make the Grid look better Your Layout Editor may appear as shown here DEIA YI ee Pe Lael De Note that any item in a Grid can be move out of the Grid or to another position just click on the item so the cell is selected and drag it toa new position 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 qa 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 gate to the Group s CD4 and CD8 gates Lesson 10 Creating Finished Reports 99 so that itis applied to all samples in W Tutorial WS cena Tutorial EE the workspace Your Workspace EMA a Sa window should appear as shown her 3 Now click on the Median CD3 statistic under the CD4 gate and drag it to th
12. command is bracketed by lt and gt do not edit any text within these brackets However you can add text between the brackets As shown below you can type in explanatory text and hit return to create line breaks to format the text box When you are finished click on OK In the Layout Editor magnify the view to 100 Click on the text box you have just created and select Get Info to change the text style For this example set the background color to gray the text color to blue and the text format to bold and centered Note that the Pen Color applies to the line drawn around the text box should you choose a Line Weight other than None When you are finished click on Set and the Layout Editor should look Dl Bevo Tet E A Layrut lten Definana Pople wea illh gn Eear ATES Daher ike Sinise Reywerd VT Rear ara Faltam K Lee pi l tee zj Careri Eras Fan Ca ber fa beg tases Toot Paia inm eat Ca br feel iz ig rivets Corie 5l JH i Thuis of iha bert trun hire oer aie for Keyword E Ein Fut biraci EEEE paree Pei To i chiki ap i feces Bhat Weir ce a e i e Feet Help Up date Layout Tarom Cat j Hein tances jf ser J 92 Lesson 10 Creating Finished Reports z jerii i a a an 2 Tutor WS End OF Tubsrial Lisit Ecitaer Chur teeter A e Ti me o g Estr OFT FlowJo Report ae os mre a aT 7 Teas iF HF ia Serin et something like that ab
13. complex as you will have 18 patient samples however you will quickly appreciate how little additional work is involved in your future analyses In the next Chapter you will learn some additional Layout Editor techniques including creating Grids and placing background graphics underneath the reports Lesson 9 Generating Complex Batch Analysis 89 Lesson 10 Creating Finished Reports In this chapter you will learn how to use some of the advanced features of FlowJo s Layout 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 ele ments 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 50 in the scaling popup menu at the bottom of the window so that you can see the outlines of the page breaks First add a backdrop From the Layout Menu select Add Picture Navigate to the Tutorial Folder select All Readable Documents from the popup menu and open the file Report Backdrop see window below This is a PICT file that was created using CanvasTM you could open any PICT
14. elements In the top panel you will create a text box containing some 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 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 T on the right side 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 Box Editing window Here you will select Lesson 10 Creating Finished Reports 91 several keywords 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 cytom eter used for collection SYS the system on which data was acquired and Cells the sample identifica tion In your own datasets you may have other keywords with important information that you can add Eval Layuul tt Keyvord 001 L ap ward SUYI ey word SUT foort value for Keyword LBeROT j Ti eet tet pract s Ta atts ceotictlo dog f fren Se Workescoe ed dra If amp ee trat B C ieo Each keyword
15. file generated by any program and add it to the layout You can also Copy and Paste any graphic from a drawing program into FlowJo s Layout Editor Get beta iteration Ophoans Fave Layout Settings Bring To Front iF EES kave Up EU Sj rw ee rF a hive Drwm Pai send to Bark li Pee miie a b i ecraitar rair vid ler LES 18 708 Griup BD Flew Pe tarkeaa Date 155 Ungroup RE TP b I F keh remeri IITA E p RRE aa sada get Grid Info ow Oj rence Testis Create New Tobbe W o o Q Teil eE Virka par Taie l mg rid ER Errem Toke Insert Picture p i TS grai Zoom In Toor Out Align EE Align Horizontal Alyn Wartical Show All Readable Dorumeris haw Preview a e 90 Lesson 10 Creating Finished Reports FlowJo adds the PICT to the layout This will be the template upon which you will add other elements like statis tics and graphics in order to create the finished report The layout window should appear as shown here For your own reports you can generate whatever design you wish including spe cific areas for text graphics signatures etc Simply design the form in any graphics program and paste it into the Layout Editor or save it as PICT and open it as you did this one Note the printable area in FlowJo s layouts is approximately 7 6 x 9 5 inches at 100 Page Setup In this report you will include three separate
16. hee ar meee CE I ee e Tirto wm she mra j a pe oy aie E Ae ane f fj i i bea h ma Er mee cer sre settee ce tee a i a o Tag Ti nay phn H ae ae inam in Ap YeEICal k Lesson 8 Creating Batch Graphical Reports Tutorial WS Ohapter Hih Layout Editor tme AERA er OE ETI Close the Tiled Report window and in the Layout Window select the original Layout that has the two graphics as shown at bottom of previous page These two graphics are a forward vs side scatter plot and a FITC CD14 vs Cy5PE CD45 plot 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 lefthand side Its name is the original layout followed by the suffix Batch It should look something like that shown above here called Layout Batch Now each Frame consists of two plots the arrow line and the text item Remember that FlowJo creates a new Frame for every sample in the current group However you may notice depending on your Prefer ences setting that most Frames only contain one plot Why Because the second plot requested a FITC CD14 vs Cy5PE CD45 graph Most samples in the current group were not stained CD14 and CD45 therefore FlowJo assumes that this graph is irrelevant for that sample In some cases however you will want to generate the FITC vs CySPE gr
17. 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 Again drag a lymphocyte gate the single popu lation only don t press the option key from any of the samples and drop it onto the CD8 subset analysis group Select the group and open the lymphocyte subpopula SS en Sampie Mise E tion from the first sample ES Sie oo rom Create a CD8 gate on the CD8 a ae RCE histogram much as you did for CD4 cells Then show the Lumpaacd ted COR 1 contour plot of CD45RA vs 1008 CD62L This plot is shown at the left 10 Move this window to the k side where you can still see Bibel it and click on the Workspace E window to bring it to the front Drag the CD8 gate I a4 you created onto the group s lymphocyte gate the my Workspace window will Ba iE 10 1m0 show the group s CD8 gate under each of the member 40 Lesson 5 Modifying Group Analyses Laat s amid coe asp tee kal RE Cyrpisoy ier ET E aiiis i Oe ka ED beprate er thr ce aA Serpe Deo Biegmmosens BD me samples as shown here in the figure to the left You will notice that the subsets of CD8 cells are similar to the sub sets of CD4 cel
18. 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 As Workspaces are the basic environment that FlowJo uses to help you organize your data they don t in fact contain the raw data Instead Workspaces have pointers to the data that you load If you move the raw data files to another disk then FlowJo will need to 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 10 Lesson 1 Workspaces and Basic Data Display 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 prepara tions from human adults Each preparation was split into 4 aliquots 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 Ex
19. patient sample Bier eT stained with the same fe A reagents is found a few cae i rows down labeled Taaa 931115 C02 Sample 02 euu ae Again we would like to gme bimi ne 3 apply exactly the same otii asa tage D fe i asart gates and statistics per formed on the first patient 28 Lesson 3 Copying Analyses to Other Samples sample to the second We could drag each line one by one to the second sample However FlowJo provides a special mechanism for copying entire analysis trees if you hold down the option key as you begin to drag then FlowJo will take the subpopulation you clicked as well as all of its descendants You will see this occurring via the dotted outline that FlowJo draws which denotes all of the analyses that you are copying Click on the Lymphocyte gate press the option key and drag it to the C02 row Even though the children of the first lymphocyte gate are hidden because you closed 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 There are more complex ways of selecting which analyses to copy For instance you can shift click several analyses to drag multiple different gates simultaneously and or you can use the control key to ask FlowJo to take the ancestors of the subset you are selecting These actions can be combined For a detailed description of these operations go to
20. polygon in SE fot 100m 10080 tO orea 7 any drawing program If you hold down the shift key you get horizontal or vertical lines you can use the delete key to remove the last vertex Either click on the first point double click at any time or press any key other than delete to close the polygon and create the gate Hold down the option key to make a rectangular gate Open the first file in the sample Workspace you created and change the graph to Forward vs Side scatter with a contour plot You will now create a lymphocyte gate draw a new gate around the lower population as shown above As soon as you close the polygon FlowJo asks you to name the subset that you have just gated see next page 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 Lesson 2 Gating and Statistics 17 success of your future analyses SSS subset Ientincation depends on having analyses with sip descriptive names For example Enter the mane ofthis subpopulation in this case you have gated lym phocytes so name this subset i Hina Holding down the aptian iy while eti eiei ay Sateet Lymphocytes Type the name E SEH into the box The Help button Heim _Select H will provide more information about naming subsets If you click on the Select button FlowJo then creates the gate and opens a new Graph window showi
21. red mama eL dots now appears to be com Ton i pletely red because the red T ee population is on top i e drawn i a last see the example on the next page Beds Segara E T 68 Lesson 7 Creating Simple Graphical Layouts o frre hati Tae BAA Ea ma Once you create an overlay graphic you can easily change its appearance axes and plot style just like any other graphic Double click on the item you will be shown the Layout Item Definition window As shown in the example below change your graph to show OrthSc vs ForSc side vs forward scatter pamje e e LJusrriernay DoR 1 j cal Empi laynut tem Detention Now the Layout Editor Window e fy arches should look something like the image below See that the dot clusters reflect the gates which defined their colors Double click on the graphic again and choose to display a histogram of CD16 FlowJo now shows you a histogram overlay like that shown at the top of the next page tzit Aera pit er ORE SS ari A jh apie Tja bapa ir Note that the legend now has an additional set of controls right next to the color boxes These are popup controls that let you set the histogram line and fill style for each subset If you click on the line control next to Lesson 7 Creating Simple Graphical Layouts 69 the red box you should see a popup menu as shown in the second graphic to the right Here
22. right portion of the Gray Statistics panel of your layout view You may Create Mew Grid have to adjust the magnification in order to see the full area of inter E a i Mambro Rows 3 est FlowJo now asks you what 3 dimensions to assign to the Grid A enter 3 rows and 4 columns FlowJo now shows the empty Grid placed in the Layout Editor see below left Select the Grid and choose Get Grid I nfo from the Layout menu Select a light yellow Fill Color Dotted for the Inside Dashing choose that the first row and the first column have headers and most importantly select the option to Force Contents to Resize With Grid and unselect the option to Automatically Resize Grid when EEEE Items are Added Unchecking this a O a last option means that FlowJo will not resize i Cale Lis ig a Serer fi Wep di rere avan ee Tel neta bite tage bared a fe a y e F T Fan Takor biii imig Dotted B finte eorna m mei i FS irmi res mrin waare Lapme E Riko ts o SS C irera be rip Brides chen ace iird Hew _tamcet set 98 Lesson 10 Creating Finished Reports the Grid when you later add graphs or text boxes to accommodate them Further the Force Contents to Resize With Grid option means that when items are added they are automatically resized to fit within the Grid Finally it is time to start annotating the Grid contents If you option double click on
23. see all of these in the ex ample workspace on the right The color style lt F 351e rph ON ie Kad cpmpaieT OF Febr BEI coe T E Dap a Piger EFi sae T E Ang TET 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 param eters etc This is how you can guarantee that the exact same analyses have been done on all of your samples We will see later how to modify Works pace hor ures jare Hide Statistics Emit Workspace Onlumns sort sample List Add Samples BEW GPU Compensate grot ps Derive Parameters Deine Calipration Kinetics Phetiri Cen Cece Matar hi Recalculate Samples Dni Analyte Edit Attributes pie inia Export _ Get Sar EH analyses for particular samples If you scroll down the sample list you will note that while the analyses gates and statistics were added to all of the samples not all have been computed Again FlowJo only computes these when it is necessary or if the sample is currently in memory To force FlowJo to recalculate all of these at once you will use the Recalcu late tree command First select the group node T cell analysis and make sure it is highlighted Now choose the Recalculate Samples comma
24. shift click multiple statistics and drag them into the Layout View they are all appended into the same text block Again you are free to edit the text outside of the brackets don t edit text within the brackets If you click on the text box with the statistic and select Get I nfo from the Layout menu you will see the Layout Item Definition win dow from which you can change the particulars of the text display shown here Select a red text color and 14 point font size click Set 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 Note that like the graphs This iza sample of the text you currently have Layout ftom Botinition Liner by Pen pAEsTeeSeseesere FIP Dobie rH i da E Eri Sele l Pen ota re ee fet pl Taxt Ekg hry eree a ame l4 4 ekart Hoa Haty Left selected Ten Cema statistics in the Layout editor are also live If gates change causing the number of events to change then they are automatically updated 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 subset that does not exist in the current Iteration sample then Tenastai Ws jih aper 3 Lyo Editer AB 84 Lesson 9 Generating Comp
25. tables Click on the Line tool and draw a line by clicking and dragging underneath the two graphics If you hold down the shift key the line is forced to the nearest 45 degree angle If you now double click on the line or select the line and choose Get Info from the Layout menu you can change the style of the line FlowJo shows you the Lay out Item Definition window as shown to the left Laytuit thei Definition ee a aooo FIM Calan Line Weight Moral Dashi Metin dah Peo tok m Arete daiken a Tit Sty he Fos ratri Fac FFM Fal 5 M Change the line to be dashed by selecting Medium dash in the Dashing popup menu and select the line to have arrows at both ends by choosing double headed in the Arrow popup menu If you wanted you could also change the Pen Color i for the line Click on the Ee e e NT TTT Ic button marked Set The es Ar poe ala dialog box will go away Your Layout Editor should A now show the dashed gt arrow and look something oy al like the one shown here sel ie bar Dasa m Bi OA Didar To create a text object click on the Text tool and then 62 Lesson 7 Creating Simple Graphical Layouts 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 a new Text Editing window into which you can type any text You can also drag statisti
26. the existing tree and add the new analyses FlowJo only adds those analyses in the new tree that are not in the old tree O Tatertal WS hapte aa on Be eae Se eee ee ETET UV epee tra Gn Lymphecyden FL PRE Sunsets T Empet ba 2 Cm lt I ainsa Same Dior m Lij SLE coke 3 Bo Mi E Dg CES i E Peay of Forest ED mz E CENS ried i Freg cof Par ent GEE a E Dyra Com Med E Freq of Parent As an example 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 popu lations Your Workspace will now look as shown to the left BD Wades E ogee coer sr see marram Here is an example where aT a you have modified the E mer me existing tree in sample wel ae B08 and wish to add the a3 changes the statistics to C08 You could simply drag the two statistics nodes all the way to the M1 subset with C08 resulting in the workspace shown on the next page Then you could continue this process However this is tedious espe cially if you have more than a few changes Instead merge the trees Click on the Lymphocyte subset under B08 and while holding the option key to drag the entire tree drop it onto the C08 sample Make sure you drop it onto the sample level that i
27. the mouse FlowJo again asks you the name for this new subpopulation subset of cells Type in CD45 as the name of the subset The Graph window now displays the gate and the statistics for that gate as shown below The vertical placement of the gate is irrelevant you can drag the gate horizontally or vertically by clicking on the blue bar the cursor will be a hand and moving it around You can change the Aii Ai Samale Bi ees Se Sl upper or lower boundaries by clicking apeese __ ARTT on one of the handles and moving it irma The Workspace window now has a new entry as shown in the example on the next page Note that the new entry is below Lym phocytes and is further indented This is because the new subset is a subset of Lymphocytes The Workspace reflects this hierarchy The numbers to the Lesson 2 Gating and Statistics 21 right indicate the fraction of events falling within the gates Thus 70 5 of the sample s events fall in the lymphocyte gate 63 of ELT aaa seer Tt pag a the events fall within both the lymphocyte gate and the CD45 gate At this time we will add some statistics to one of these subsets Click on the CD45 row so that it is highlighted and then click the middle button in the button bar the Sigma button This indicates that you want to calculate a statistic of the currently selected subset You will now see the Statistics window as shown to the ri
28. the on line help documentation and find the section about dragging and drop ping analyses woke Again you will see that FlowJo has not actually ace ah calculated any of the analyses you requested If baat Workspace Cohamns f Sort Sample List you wanted to see the results right now you can an 7 force this calculation to occur Click on any pop How Greup a6 i c ate sampe ulation and select Recalculate from the A Workspace menu Note you can also select a Define Caliwration Preference that will control whether these recalcu Pen i pa aa lations should always be performed immediately The Recalculate command tells FlowJo to calcu late the gates for the currently selected node and to perform all statistical and gating calculations for any of its children Select the new lymphocyte subset and select Recalculate from the Workspace menu or press FlowJo reads Det Sample btw Export Lesson 3 Copying Analyses to Other Samples 29 in the data file and makes the necessary computa tions The workspace now appears as shown You can delete analyses by selecting them and sie ie eae pressing the delete key Select the lymphocyte subpopulation that you just created and press the delete key FlowJo asks you to confirm your choice telling you what the consequences will be below Note that there is a help button clicking on this will take your web browser to help specifically about deleting nodes
29. the right za of the new Lympho wran cyte subpopulation E depending on the r current setting of eee memme mae _ Es TO FENIT terete iD fon 1 L eeiesi COA TE ie your Preferences FlowJo does not in 26 Lesson 3 Copying Analyses to Other Samples Fitch hic Soh sit dL fan Tek 1 Fie aa Tree ai ELA epee ya ie F K 1 fF BE on a a i r 4 1 i i i i tl i a T T L io loa iin general calculate statistics such as frequency until they are absolutely needed the display in the Workspace is not considered a high priority Later we will see how to override this behavior In any case you can still double click on this population to open a graph of the lymphocytes within the 931115 B02 Sample 01 file Now open this graph and choose to view a histogram of CD3 Create a gate on CD3 cells and name it T cells At this point your Graph window should look like the example here top left Click on the Down arrow to open a graph of the T cell subset Create three rectangular gates on the CD4 single positives CD8 single positives and the CD4 negative CD8 negative dou ble negative T cells as shown to the right In the Workspace window click on the triangle next to the very first line These triangles open and close the views of the subset hierarchy much like they do in the Macintosh Finder In the genealogical terminol ogy tha
30. 14 Create two gates for the major popula tions that you see This time press the option key while you click for the first time to create the gate FlowJo will restrict your gate to be rectangular Rectangular gates will be com puted 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 both gates to be rectangular see the graph above Name the upper gate CD16 CD14 dim ProMono although this may well be granulocytes you could name it Granulocyte 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 on the next page Note that the Monocyte subset is indented one level as is the Lympho cyte subset Both populations are subsets of the sample The Mono and ProMono subsets are indented to a second level and are shown below Monocytes because they are subsets of monocytes not of lymphocytes This hierarchy of this gating is visible from the Workspace window 24 Lesson 2 Gating and Statistics This hierarchy is important in that if affects how FlowJo works It is analogous to a genealogical tree i Seve kt far in that each subset
31. Copy from the Edit menu or press C You will 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 You will now create two more for this workspace To create a new layout click on the New button near the top left of the window To delete a layout click on the Del button Click on the New button and name the new Layout Scatter Analysis Now select Paste from the Edit menu or press V the previous graphic will now appear in the new layout Paeria Ws Shapter 7 Lavwut Editar fet FE BCTI j E ue i EE sevar Jona We will use this particular layout later For now let s create a third layout to demonstrate how to create overlaid graphs Create a new Lesson 7 Creating Simple Graphical Layouts 65 layoutandname G menats chapter 7 Leyout fier BES it Overlay Drag FEB e Beng uae riers Le the first Lympho r cytes subset from 931115 B01 and drop it in the view You should have a view of the CD45 histogram as shown here Double click on the graphic and change the graph specifica tion to be Dot Plot of CD14 vs CD45 The Layout Item Definition window should appear as shown below Click on Set and the Layout View now shows you a Dot Plot as shown on the facing page Layout Iteni Definibian Sample ed eet eee ee Be Papalit fea Lyp bet aroctats S
32. FlowJo Data Analysis Software for Flow Cytometry User Documentation Tutorial TREE STAR FlowJo was written by Adam Treister and Mario Roederer based on concepts developed with David Parks Martin Bigos and Wayne Moore We are indebted to past and present members of the Herzenberg laboratory at Stanford for their ideas discussions and tireless testing of new versions Mercutio MDEF is Copyright Ramon M Felciano 1992 1997 LMoovController is Copyright Paul Lalonde 1998 Various utility code was written by John Norstad Northwestern University FlowJo is Copyright Trustees of Leland Stanford Jr University 1996 97 FlowJo is Copyright Tree Star Inc 1997 2001 FlowJo Tutorial and Web Site are Copyright Tree Star Inc 1997 2001 Revision Date 18 April 2001 Version 3 4 FlowJo Tutorial FlowJo is a software application designed to create an integrated envi ronment for viewing and analyzing flow cytometric data This environ ment 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 lay out 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
33. Jo compiles all of the graphs statistics text grids and backgrounds and puts them into a single window see below From here you can print a report publish to the web or copy to slide presentation programs io i Tia Ei Diiin Fiii j apart FlowJo Report FlowJo Report Most importantly your Sa layout is now ready for ge m the next experiment all you have to do is add the samples to the Workspace and run the Batch proces sor 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 This ends the tutorial lessons There is more documentation available a En in the reference web MOTURID pages which you ll reach from any of the Help buttons within the program by typing anywhere in FlowJo or by looking at lt http www flowjo com v3 reference html gt If you have any questions or ideas for improvements please contact us at flowjo treestar com Lesson 10 Creating Finished Reports 101
34. Lesson 8 Creating Batch Graphical Reports 77 If you select Show Breaks then FlowJo will rescale the page size to whatever you have selected with Page Setup If you select Avoid cut offs then FlowJo uses your Page Magnification but places frames on the page such that frames don t cross page boundaries unless a frame is larger than a page Use the current drawing to experiment with the three options that affect how graphs will be printed 1 the layout dimensions using the Layout Specification popup menu near the top of the window 2 the page dimensions using Page Setup and 3 Page break control using the popup at the bottom of the window Once you have arranged the Frames exactly the way you want you can print them you will know exact ly how they will appear on the pages It might be cumbersome to have to redesign the printing layout every time you create the batch out put Therefore FlowJo lets you save the current state of the Batch Output with the Layout simply select enmet Save Layout Settings from the Layout menu Align Horizon i see right or save the settings when the batch report is created Either method saves the values of the Page Setup magnification and orientation and the setting of the Layout Specification popup menu i e how frames are to be placed in the overall output the page break and scaling specification 78 liana ph apnEr Ay laur fatar 2B Sood Bet Deter
35. 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 subpopula tions for the first sample are drawn in black this is because you modi fied them and they are no longer equivalent to the versions in the group However these are the ones you want to apply to the group Once again a eel arie ale this is easy to do shift click the four subpopulations Naive M1 M2 and M3 under the first sample and drop them on the CD8 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 green since all are now identical see right You are now finished with the T cell analyses go back to the PBMC subset group Shift click the Lymphocyte 42 Lesson 5 Modifying Group Analyses and Monocyte gates from the first sample and use the option drag action to move the tree to the group node This applies the analyses to the entire group see right Now if you click on the Experiment 1 group you can look at all of the samples from this experiment in their full glory below Note that the assignment of color and style to the groups helps you identify which group any particular gate comes from As a final tune up for the gating analy sis you will modify the lymphocyte gate and update all of the samples for this experiment to have the new vers
36. 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 opera tions such as creating new graphs statistics 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 software compensation exporting raw gated data for analysis by other programs 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 such as Netscape 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
37. Ree Ti te D popio a EEIN 1 es Tote SER DH pk TE ie EP kaetregter SP Pera BR Dog ras he te SS cpi wa E Tie coer 2 fem af Pareet Workspace menu If you invoke this command after clicking on a sample s subset then only that subset is updated to have the same gate as the current group s version of the gate If you invoke this command after clicking on the group s version of the gate in the upper half then all samples in that group are unified So click on the Lymphocytes subset under the Experiment 1 group and select Unify Analysis FlowJo asks you to confirm this action see below If you click l Contre action Oo abu wie all cae pee ler thee giia he baltic versions of selected gates Hiki m be ear ort ree ae bar E Lesson 5 Modifying Group Analyses Workspace Roe Watrks gph H Hide Statistics p Edit Workspace Columns Sort Sample List Adio Saiples a Mow Group it Compe nes abe rr 3 herve Parameters b Beime Caliah Kinetics Hatitrm HK Cel Cyrie Matt erm Recalculate Samples Se Unity Analyses pe Edit Attributes xA Got Sarmah biti Export 45 on the Yes button then your Dos teeter Workspace will soon look like i the one here at the right PINE abarir Note now that all Lymphocytes T Laapieytas subsets are drawn in plain blue F EE text denoting that they are identi Deea EE Hererg Sen cal to the group s version Open S511 007 Hngh Oa a
38. age 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 some thing 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 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 sample 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 speci fied by that group as long as it is applicable to the sample This will become more clear in the next few steps of the tutorial This lesson buil
39. ample the graphic on the this page shows the result of copying the above table into Microsoft Excel The third button prints the table and the fourth button saves the table on disk and immediately launches an application of your choosing to analyze it The length of the column headings often results in long names as exemplified in the Excel spreadsheet 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 In addition there is also a preference to Use Short Stat Names in the Tables Layouts section but usually you ll be able to come up with more descriptive names than the defaults provided by the program 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 Click on New and name the new table T Cell Subsets From the 54 Lesson 6 Tables and Layouts Collating Data Output Workspace select the T Cell Analyses group Use the option drag action and move the Lymphocytes subset from t
40. an name them M1 M2 and M3 Lesson 4 Groups and Batch Analyses 37 0 ST ah a a ties Your graph should now show these four gates Close the Graph win Peeves isis f 0n tse e eae dows If you hold down the option key while clicking on the Close win dow box the top left box in the Graph window then FlowJo will close all open Graph windows Your Workspace will show the subsets you just created including the differenti ation subsets of CD4 cells which are a subset of lymphocytes which are a subset of the entire sample see below Lyrepresgiaa Gott 7 DOO Feen F Note that the CD4 gate and the CD4 subset gates are not in green This is because these gates have not been applied to the group and therefore are not identical to a group analysis They are drawn in plain black to denote the fact that these analyses are unique versions belonging to the sample D i mmeanaWwsihater y OB AMAHA To apply them to the group click on the CD4 T subpopula w led ai tion and use the option drag Mij capartinart action to move the gates onto nida the Lymphocyte subset of a the group Note that if you TT ANETT tenet 0 eo raa ih were to drop it onto a higher ener iat ae level a group name the tree aa a would be applied to each Da aE iae sample at the sample level i ee bam esi not to the lymphocyte gate ED yong t That is the gates attach them n ee eee s
41. aph irrespective of what the actual stains were FlowJo lets you over Lesson 8 Creating Batch Graphical Reports 79 ride the stain specification Select Preferences from the Edit menu and click on the Layouts tab FlowJo shows you the following window that sets a variety of preferences related to Layout generation Click on the checkbox marked Allow Stain Name Mismatch Save the preferences and then regenerate the same layout You will now see the pair of graphs shown for every frame 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 a one of these reports and then change a gate the report is NOT UP DATED You will have to regenerate the batch output to reflect the change you have made Yio aCas G General Tabie Duipuai Yii Leymus Apperasces O Sins Summary Siats O use Short Stat hames Urap ress Axes Labela meit Ford Leyeuts Behavior m tating Baish Format Ei a syu m Offset Histogram Over les Graphs Fr eguemcy tect Egue Stai Mame Mismatch O Use Placwhokdars Fort Pata ines C Ceto ees Humi tem Settings disieg Si Ea aj ACE T bef e Layoal Annotation tanotation Box Defaults Lateran tan Asmo Pauitin ame pory raa O a or EA Stew Sample Hare C Stew Full Patt te O Stew F
42. c into the Layout Editor and drop it releasing the mouse button to the right of the graphic FlowJo creates a text box the contents of which is the name of the statistic and the statistic value as shown in the figure You can add addi tional 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 click E a Tutorial Wi Sieh asi CECERR p ihe ol Saretes ie Subraes ios Subset EF PE tD wer oan Seance 0 er EESE ee py E CpSPE CDAS CY E cysred CD45 Had 5 Freg ef Grandparea Ereg ef Parent Pz Piteeiies GD Mion E Freg of Parent ED prerana Freg ef Parent Se Lopta Editar acd um races Tins walk ing on the lower right handle and resizing it to a larger size Then double click on it to start editing FlowJo opens a new 82 Lesson 9 Generating Complex Batch Analysis _ JE Edit Layout Text Window O Fatt Layout tot _ as shown to the right Friaj of Parant Statii joo 2eassd57 Lempherwies CHa T reg af Pare Note that the statistic value has been expanded to a bracketed lt gt command displayed in red The command with in the brackets tells FlowJo exactly how to get the desired statistic i e which subset and which statistic is desired Do NOT edit the text within the brackets or else FlowJo may not be able to fetch
43. choose to show the Legend box for any graphic even if it is not an overlay from the Annotate pref erences in the Layout Item Defini tion Window Then you can set color and line styles for single graphs just like for overlay graphs Lesson 7 Creating Simple Graphical Layouts 67 TRL eer EE L T As noted before all Layout Graphs are live This means that if you change a ETET parent gate for one of the subsets the graph is automatically updated To see this in action switch your attention back to the workspace and double click on the 931115 B01 sample node You should see the graph to the right defining the gates for the Lymphocytes and Monocytes subsets Click on the Monocytes gate upper right gate and move it around perhaps as shown in the second graphic to overlap with the Lymphocytes subset are belhamel hh m Elina i e ee A Deen ays ja 118 Chee re Note that the Layout Editor responds by updating the blue dots corresponding to Monocytes 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 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 Lym phocytes subset and move itto Sia i Sas the top Note that the area Ee ee ee which has both blue and
44. cp Set the iteration value back to Off This specifies that FlowJo will only show you the graphs and statistics from the originally dragged subset The graphs and statistics shown in the Layout View were derived from the first antibody combination CD14 CD16 CD45 Now we will drag in a graph from a different staining combination Select the T cells subset from the sample 931115 B02 and drag it into the Layout Editor next to the first graph Your layout should appear as above Add a text box item above the second graph typing in T Cell Analysis Select the text box then choose Get Info ae from the Layout menu PETER re bee tes Taree E ETE and set the font size to 36 SARA will look something like that shown here point The Layout View ator e i Make the Layout Editor a T 8 Window larger by drag on a ging the lower right ail hand corner to resize the Shas Lesson 9 Generating Complex Batch Analysis 87 window in order to make room for e Tmn n e Ga more graphs in the Layout View Backin EAE the Workspace Window click on the CD4 T subset and shift 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 a ae rare 3 ee dee TE ra eat rc A ar ay agi T ie wwe lie E fal pa EH i E
45. cs from the Workspace and add Keyword values by clicking on the Keyword menu These items are live and will change as you create batch layouts for P Hit avout lat Fe multiple samples Farncimias Si Type PBMC Analysis into the window and click on OK You can change the font amp style of the text ina kort rahe for Keyword Saket eat FIL rajy text box by selecting the To ade SARS dag AF prose fie Borka and dop AF i the tert text box and choosing Heie Update tavoit cancer om Get Info fromthe Layout menu Note that if you double click on the text box you will go into the Text Editing mode where you can change the contents of the text Select Get Info to change the text color and font size as shown at left Select the Text color to be red and the size to be 18 Note the fill color is applied to the whole bounds of the text box the pen color is applied to the box drawn around the text box which Layout Rem Definition is drawn only if the Line Weight Caer Lie Style is set to something other than Haley e a None Click on Set to confirm Ferner a sian the changes EET eaen 5 matin Your Layout Editor should appear 2 En Lan i as the example on the next page Lesson 7 Creating Simple Graphical Layouts 63 Tutorial WS Chapter T Layout Editor w El Maree Diin Plein leer EIEE moki R bii imei Hi i Eviran Kiih Layout
46. ct the HER iteration popup menu from 937ht8 B07 Sangle Dt fer the Layout Editor window z Lyppherytes you will only be given a EAA selection of four different Sanpete samples to choose from see below Again this is because whenever iteration is not off FlowJo looks through the current group to decide what possible samples can be dis played Note that if you construct a Layout for a specific group which has unique sets of gates and statistics ES then the layout may TE EI A E Eare DI iea n Eaa Saary not operate as desired on other groups which don t have those gates Once again if you select Off for the current iteration value then FlowJo shows only the Ea original graphs for this layout View the ones you 74 Lesson 8 Creating Batch Graphical Reports 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 Placeholders checkbox is not checked 2 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 3 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 Lymphocyt
47. ds emph im iratber peep hr Dwy diina frar rehat tes chee al Sees Bee Oe eee ree Cot a a or eee a oe eee ee oe te _Done at the top and click on the Create 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 SSPE city Gra Lip ATRL Ee E rgap Displey Are Fr per jie en Jespla iecissian Critorin Bey ck ei Da o ir air I wa TE GS TL Kiely bird had Cider ee Thad ete Cire bebe kig Ca ela Oe Coe a oa oH Laceliectin CES E Lobehn Ca oe Gaig ieaiai Balimi Here men menghed thit baei Wilk refergecy Ge pamplap in maribor grea EMADE EEN Ep Gre We R E BB aha miea g Soe Oise hire create your second group corresponding to the analy sis 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 from the Style popup menu Your window should look as shown to the left Lesson 4 Groups and Batch Analyses 33 marai WS Chapter 4 BI Now click the i Sewers ea Create button to create this group Finally make two more groups called CD4 Sub sets and CD8 Subsets adding the third and fourth stain combi so E page nations respec TIBADATZ Marais Eeg a tively and set the ala ne F color to g
48. ds on the Workspace you finished in Chapter 3 Or open our pre made 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 antibod ies CD3 CD4 and CD8 In the Workspace click on the second button in the button bar Create New Sample Group FlowJo brings up the window shown on the next page Using this window there are a variety of ways to make new groups In the middle portion of the window is 32 Lesson 4 Groups and Batch Analyses listed every different combination of stains present in the whole list of samples They are pre sented 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 sam ples with this combina tion Enter the name T Cell Analysis in the box 5 jerii Groep Atbriietes Ta oe pram Gey ele __ _ ___ ____ ___________ tore Ete wake d rs in Cre Sample ben l aiien iriteris EA ates apply criteria t all rarer eph mallee a wert Lien qraoph Deb wehiy pare les Wet ase Ikr foley iar beg is te Co an os SH Landen Cee ond L ren Cae Ee Thy japhnia samples haf hrar akei rikr mper Wih cefercere
49. e or select the Table Editor from the Windows menu FlowJo shows you the Table Editor window and automatically the first table change its name to PBMC Subset Freqs as shown here Click in the title field and type the new Tutorial WS Chapter DEJ Table Editor Ei name for this table E ew Par weir era In this window the left side holds the list of existing table templates The right side will show the items in the current ly selected template 50 Lesson 6 Tables and Layouts Collating Data Output What is a template When you define a table you will add rows to a table template Each row 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 re quests the particular statistic defined in the table Then 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 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
50. e second column of the second row of your new Grid drop it when that cell is highlighted FlowJo creates a new text box with the statistic and adds it to the Grid Add the Freq of Parent statistic to the next column Repeat the two drags for the statistics bound to the CD8 subset into the third row of the Grid Select the four new Grid items and change their font size to 9 If you wish you can double click on each item individually make sure that only that Grid cell is selected in order to edit the text remove the anno tations from the Text to leave only the statistic for each cell oo o To add graphs to the Grid click on the population of interest from the Workspace and drag it into the correct cell of the Grid Or if you ee already have the graph in the Lay ii A 7 out Editor you can drag it and drop E it into the Grid Select the CD4 subset and drop it into the last column likewise for the CD8 subset To change the graphs to histo grams of CD3 select the two cells with graphs and double click Change the X Axis to Fluor and the graph type to Histogram At this point your Layout Editor should look something like that shown above a ae 100 Lesson 10 Creating Finished Reports Now you are ready to generate the full report for all samples Remem ber to select the All Samples group first any batch layout operation is only performed on the current group and click on the Batch button Flow
51. e click on the first line in the sample list you will see the graph shown right 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 Thedefault plot that you see the first time you open a file is determined by the Pref erences setting Select a different default plot by going to the Prefer ences dialog under the Edit menu Note that this selection only applies to workspaces you create subsequently it doesn t affect the currently open graphs The easiest way to change the graph is to click on the information button with the encircled i at the top left of the Graph window This button brings up the Graph Tools shown to the right Here you can select the parameters you wish to view on each axis the kind of graph to plot and some of the options that affect the presentation of 14 Lesson 1 Workspaces and Basic Data Display 991 TEN Sample Olies se ah E Eems i000 woo 100 7 Axis Definitions x Axis OrthSe oe Plot Type amp Options Graph Type Contour Plot Contour levels 5 Probabil amp A Smoothing J inverted O High Resolution O Show Outliers gt Vertical Scale Histogram CDF gt Gate Options the graph Change the X axis to CD14 the EN Y axis to CD45 and the plot type to Pelee o ae ties Pseudocolor Click the Apply button in t
52. elves to the row that they are E Ne 38 Lesson 4 Groups and Batch Analyses dropped on Your Workspace now shows these gates applied to all of the samples in the group as shown here As before you can tell the program to recalculate the new populations and frequencies or leave them as is and FlowJo will calculate them automatically the next time you view print or export these populations 3 an Samples S Eo Subset v ED Lymphocytes v amp cos T D mi amp m2 ED mz Ae HEME BS Statistic cens wv 931115 B07 Sample 01 fes v G Lymphocytes v amp cos 7 ED m amp m2 m amp Naire Vv 931115 C07 Sample 02 fos VD tymohooytes_ v amp coe T gt mi m2 E mz amp Naire 7 931115 D07 Sample 03 fcs kag amp Lymphocytes v amp cog T ED m amp m2 GS mz z Lesson 4 Groups and Batch Analyses 76 78 45458 2 29 3 478 148 6 49 16 A bd S a E z 4 39 Lesson 5 Modifying Group Analyses In this lesson you will learn how to take advantage of previous work you have done drawing gates generating statistics to save time when you do similar 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
53. en which for this dataset con within a Cinque Heratica tains the unique patient ID When you click on OK FlowJo will now search through every sample in the current Group and build a list of unique values of the Keyword Cells found in the current group This list is shown in the Iteration Popup menu you can see it by clicking on the a Taai a W espe hy Larit Edino pg menu shown here at right EAP e ape ene n E e ees tt Note that the title for the Popup menu has changed from Sample to Cells to let you know that the Attribute for Iteration is now different In the list of all of the samples in the current workspace there were four unique values of Cells When you now select any given value of the iterator then for each graph and statistic in the Layout View FlowJo searches through the current group to find the first sample that 1 has the Keyword value corresponding to 86 Lesson 9 Generating Complex Batch Analysis the current Iteration setting 2 has the subset specified by the graph or statistic and 3 has the appropriate parameters for the graph and 4 has the correct stains defined for the graph Criterion 4 can be overridden by the Preferences as explained in Chapter 8 However you should in general not override this Preference if you are perform ing complex layouts like this since your data is surely correctly anno tated already fie WE eer hs oe ee eee aes e
54. ental name by which FlowJo identifies subsets of cells Lesson 2 Gating and Statistics 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 gating to create a subset or a statistic and you drag it to another place in the Workspace 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 alterna tively you can open the workspace named Tutorial WS Chapter 3 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 lympho cyte 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 row is highlighted then let go of the mouse FlowJo creates a new subpopu lation using the same gate you had previ ously created Your Workspace window now looks like the one shown here Ss Note that there are no Beye numbers to
55. entially the equiva lent of dragging the tree to every sample in the group and choos ing Replace Fe baste Freg of Puree eee Cg SPE CS MEL Treg oof Ppi 7 F L ea COS Hie Frey ot Rarer are Cy SPE Cid ii E E T es E ze n E E CSPE Cie Mied Faj ai Fagat F Cy SPH Chet raed Lesson 5 Modifying Group Analyses 49 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 Editor you can click on the fourth button in the button bar it looks like a tabl
56. es then FlowJo looks for a CD4 subset of Lymphocytes in the current sample These two criteria cannot be changed There is a fourth criterion that can be overridden by the Preferences and that is 4 the current sample has the same stains like CD3 or CD4 for each of the parameters as does the original sample In the Preferences under Layout you can relax this constraint This may be necessary if for example you have not properly annotated your samples Mieke baici Report So far you have only looked at the Layout Outpt Biel eed rere te ore ee ete of fore Report for individual samples one by reir hing dierent maya wf preranim the Bria one To look at graphs for all Bisa VA samples at once click on the al ney Ee Batch Generation button left Plight haar dang ee een which looks like a stack of papers us vuetenecs furscst ecg next to the Iteration Popup menu This Cr aaan eee eae dialog will let you set the options govern maiia pua ing how the report will look You can set Pike Taniien the format of the report the group that E empi will be processed the geometry of the Dpines rows and columns in the report and PETAEN P A other options to set FlowJo to do the EE EE EE iy ernie right thing without asking you every any a CE Hele anced i create Lesson 8 Creating Batch Graphical Reports 75 time Select the Tiled Report set the geometry to 4 columns and click on Create When you generate a Ba
57. esess 40 Lesson 6 Tables and Layouts Collating Data Output 0 50 Lesson 7 Creating Simple Graphical Layouts cccccccecesesesseeeeees 57 Lesson 8 Creating Batch Graphical Reports cccccsseseseseseseeeeeeeees 72 Lesson 9 Generating Complex Batch Reports cccccceceseseseeseeesees 81 Lesson 10 Creating Finished Reports cccccccccseseseseeeteeeeesseseseneeees 90 4 Introduction This chapter is designed only to give you a quick overview of the power ful 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 de signed 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 Tutorial Folder on your Macintosh 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 and a folder containing the Tutorial data it has within 2 folders of experimental data collected on different dates Locate the workspace document named Demo Workspace and double click it to launch FlowJo terial Merieer Envy
58. ew 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 holding down the option key while you click and drag that item 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 The easiest way to create a table in the Layout Editor pe 7 is to drag a table from the Table ni fae Editor and drop it in the premier Layout Editor Open the ba oD ypwiy en Coicta n Table Editor window and a erect eae click on the table you previ i laipa Taio ete oyani imiss Eok Eie iit i Eiei ously created T Cell Sub sets Now drag this table Lesson 10 Creating Finished Reports 95 onto the Layout Editor letting go of the mouse when it is over the gray Statistics panel FlowJo automatically creates a Grid item filling it with infor mation amp statistics from the Table Editor In FlowJo a Grid Item is sim ply a collection of containers that hold other Layout Items In this case the Grid Item contains a series of Text box items each with either static text titles or with Statistic commands Click on one of the boxes in the grid it will be highlighted in light blue then double click on the text You can edit it however y
59. f tie same names Ketsin Do eet modify existing analysis gates if they have ihe same same Rename Headity the name af the new analgais trees be adimg a version Gime Cantel Rename il Retain C repiace Lesson 5 Modifying Group Analyses lost and the resulting analysis tree would be identical to the one in B08 In this example since all of the analyses in C08 are group analyses they are identical to the analyses in B08 so in this instance Replace and Retain are essentially the same Choose Retain The Workspace will now reflect the newly merged trees below This is how you can easily add any modifications to an analysis tree from one sample to another simply drag the whole tree You can then choose to keep the Retain option or throw away the Replace op tion any differences between the common parts of the two trees You may have noticed that the Retain or Replace dialog win dow did not appear when you dragged analyses to a group even when they already existed for example when you were modifying the Naive M1 M2 and M3 subsets for the CD8 analyses at the beginning of this lesson FlowJo assumes that when you drag an analysis to a group you want to replace all of the existing analyses that have the same name Then every sample that has the group s version of those analyses will also get the new version that esse you have copied to the group a Dragging an analysis tree ontoa_ group is ess
60. ght dH Hee On the left side of of the Statistics window is a list of the statistics available Some of these statistics Fal wiatiolic i require that you choose a parameter Fig aaa 1 ramos on which to calculate the statistic for oo E i e example Mean fluorescence If you wish to compute a specific percentile of one of the parameters 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 CD45 select the parameter from the Channel 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 close the window Once you close the Statistics window your Workspace window has several new entries as shown on the next page 22 Lesson 2 Gating and Statistics The new lines begin with a Statistics icon You can eee click in the middle column Tree name bar on vertical dividers and drag to resize columns to show the entire names The statistics icon is followed by the parameter on which the statistic is computed if applicable the name of the statistic and the computed value The median Cy5PE CD45 fluorescence of the CD45 Lymphocytes is 114 the distribution of CD45 has a coefficient of variation of 33 The Freq of Parent statis
61. he first sample into o Tutorial WS Chapter 06 Table Editor H amp ES PBMC Subsets ED Lymphocytes Frequency EB T Cell Subsets D Lymphocytes T Cells Frequency D Lymphocytes T Cells CD4 T Frequency Lymphocytes T Cells CD4 T Freq of Parent Lymphocytes T Cells CDS T Frequency E Lymphocytes T Cells CD8 T Freq of Parent amp Lymphocytes T Cells Double Negative T Frequency Lymphocytes T Cells Double Negative T Freq of Parent the Table Definition window you will see all 8 rows Create two more tables for the CD4 Subsets and CD8 subsets Again option drag entire analysis trees to bring everything into the table Note that you can change the order of the table entries by clicking on any entry and drag ging it to another place You can also delete a table entry by selecting it and pressing the delete key After creating the last table the Table Editor window should look similar to the one shown on the top of the next page 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 You can now apply these table templates to the appropriate groups and get output tables with specific sets of statistics just select any group and click on the Table button Lesson 6 Tables and Layouts Collating Data Output 55
62. he next page are examples of some of the other types of plots sup ported by FlowJo Lesson 1 Workspaces and Basic Data Display 15 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 envi ronment across analysis sessions ES a a arpha ies A ee ee E Contour plot with outliers te ta Emar a an iem Smoothed pseudocolor plot Histogram plot 16 Lesson 1 Workspaces and Basic Data Display Lesson 2 Gating and Statistics In this lesson you will learn how to gate on 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 represent a subset of the entire collection with specified properties e g Lympho cytes 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 just click inside a graph when the mouse appears as a cross hair and move the mouse Each time you want to change directions click and continue to drag This is the same process as er atone aaa evel alk you would use to draw a
63. he top right corner of the window to activate the changes The data graph will now look like the one to the right Another way to change the param eters on the graph is to click on the axis name a list of parameters is shown in a popup menu _ TETI For example to change the Y axis to show Side maan q scatter click on the Y axis name box and select OrthSc Orthogonal scatter F 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 4 change individual options by selecting them in 2p the Graph Menu Try deselecting High 1s Resolution for the pseudocolor plot this type of plot low resolution pseudocolor may be the best for slide presenta tions 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 t
64. hocyte gate to the Experiment 1 group Now look at the Workspace shown on the next page It may not look as you expect sako es mel mom mas ao ee eee ab O Tii A hari o Be First note that the lymphocyte gate that you dragged is now light blue plain type this is exactly like the Experiment 1 group However none of the other Lymphocytes gates are drawn in this color or style Therefore they are not the same as this gate Open another sample to convince yourself that it F ata a ai hasn t changed Why Because of Fur the second rule of group analyses ae AR AE when you modify a group s analy as EG aes sis or add a new analysis to a 44 Lesson 5 Modifying Group Analyses group then all samples receive that analysis only if they don t already have an analysis of the same name All of the samples already had their own Lym phocytes gate Therefore FlowJo did not replace or modify them This is a very important feature of FlowJo this feature allows you to perform group level analyses but maintain individual sample variation You can modify a single sample s lymphocyte gate and still have that subset behave like every other lymphocyte subset Let s say you want all of the samples to have the same gate FlowJo gives you an easy way to force all analyses to be identical You do this using the Unify Analyses item under the 14der aed ETIN IET
65. how how many data files are included in that group You can resize the Workspace window as with any Macintosh window you can resize the group and sample portions by clicking and dragging on the dividing segment 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 S ba tonics peer Mi pl aye Perrie zaiona velar Colores lu dinpsg Theo eS T oy 0 tad Coie ot fl apa ry Scie aac e a eee __ terete You can add additional columns to the Workspace display that show the stains used for each sample or any other keyword found in the FCS datafile You may also sort the sample list on the basis of any of these values To add the reagent list to the workspace view click on any sample and select the command Edit Workspace Columns from the Workspace menu FlowJo shows the window above Move the desired column headers from the list of available attributes on the left to the list of visible attributes on the right Click Done to accept the changes and the Workspace view will add the additional columns to the table Sens rhe tance i Tae j suena i at Tae E id IENA E4 To view the data for Epin TAR jiri any sample double ee a click on one of the Neo ee 8 ee oles Lesson 1 Workspaces and Basic Data Display 13 lines in the sample list When you doubl
66. ion 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 very first sample at the top level the sample level 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 top of the next page Move all of the handles in to make this gate smaller The Workspace window will now reflect this modification by drawing the newly Lesson 5 Modifying Group Analyses E frees qf Foret T FEE dare En TEE T p E teqorv cea ce E teq wre cota Fii G im Se ierg ai Farmi n E A gegen gt Si Care G Yal Gia oy E Cy gPC CHT Mir T Prag of ais mirai m ASEET Sample Mes amp modified lymphocyte gate in CCIE Dy ee SESE lee black See the second line in Fejensix the figure below F d t How can we update all of the samples for this experiment All of the samples for this experiment belong to the group Experiment 1 the group that was created when you read in the folder of FCS files So you can use this group for analyses that apply to all samples Alternatively you could create a new group select all samples and drag them to the new group to add them and then use that group Drag the modified lymp
67. is Sipen like a child of its E taski an Cs 1 ti i pauna beiin arent population By ot irweipar wri P P p E eh yi Pereri Thus in this example l i SD riger Hera Lymphocytes is a E trea of rei child of the sample fg ea and a Parent of eee CD45 and a sib ling of Monocytes FlowJo does not allow you to name siblings i e gates on the same population with the same name 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 fundam
68. is a set of controls near the lower left of the window that control the magnification of the view You can scale to any percentage or select Auto which scales such that the entire batch layout can be seen on the screen see previous page In this view FlowJo is drawing four columns of Frames This option is controlled by the Layout Specification popup menu immediately to the left of the Help button Here you can select any number of columns between 1 and 4 or you select Custom and define exactly how you want FlowJo to arrange the frames on your page You will notice that FlowJo draws gray lines in the window these correspond to page breaks were you to print the 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 magnification Changing the print magnification and orientation landscape or portrait can be easily done using Page Setup from the File E aaa Ee ee eee menu and setting the magnification you want You can also specify that FlowJo automatically scale one page to be as wide as the graphic display This is done by selecting Scale To Page in the popup near the bottom of the window right When Scale To Page is selected the page magnification but not orientation from Page Setup is ignored oe E
69. le was named by the date of collection 931115 a code related to the antibody panel B C D or E and a sample identifier e g Sample 01 Just by adding the data files to this workspace FlowJo has already accomplished most of the analysis steps for this data Each file was examined to see what reagent panel was used to stain the cells Depend ing on the panel FlowJo added the data file to one or more Groups Then the gates statistics or other analyses specified by each group were automatically added to the sample Thus gates and statistics specifically designed for each reagent panel were applied only to the sample tubes for which they were designed You can scroll up and down the sample list to see the variety of different gates and statistics that were applied to each sample Note that FlowJo has not yet computed the gates and statistics you could request it to do so if you wanted right now However the default behavior is to compute them only when needed for the generation of tabular or graphical reports as we will do momentarily This workspace also has several graphical layout reports saved with it FlowJo saves not only analyses gates and statistics with workspaces Introduction 7 but also any tabular and graphical reports compensation matrices kinetics amp cell cycle analyses and calibration standards Everything you do is recorded and saved so that you don t have perform the same steps
70. lex Batch Analysis 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 re ports 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 infor mation to your data ask your local Flow Cytometer operator or Instru ment Sales Representative For the demonstration in this tutorial the data supplied has a Keyword Field Cells which ha
71. ll also see that the Up arrow button is now active Clicking on this button opens the graph you 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 new Graph window for the subset see below This graph is not particularly interesting since it shows a subset of the same data as the previous graph Change this graph to CD14 vs CD45 your graph now will look like the one to the right Note that most of the E ae a ye E events are CD45 positive and CD14 aai ETOR E negative this is exactly what we expect for lymphocytes in PBMC The CD45 figejeciee 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 bring the parent population visible You should be able to see both graphs simultaneously Plier S FlowJo uses dynamic recalculation of 1 i ie Om gates This means that whenever you Lesson 2 Gating and Statistics 19 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 hand If you now click and drag you will move the gate Move the gate so that it is over the monocyte population as shown lef
72. ls So go ahead and copy the same gates you used for the CD4 cells onto the CD8 populations you will then fine tune them 0 SSS Dib CG Dran tamplrs i rs ra k Tii 5 Rate First select the CD4 group again Now shift click on the Naive M1 M2 and M3 subpopulations from one of the samples see right Drag this set of gates and drop it onto the CD8 T gate in the CD8 subset analysis group De Sea age are eT e E i ery LETEN x L L Watch the open Graph window as you do this the four gates will appear in this window as they are attached to the sample Again FlowJo makes sure that every window that is open will reflect any changes you make The Graph window appears as shown left It is apparent that the gates are not posi tioned properly for CD8 T cells these are the gates that were appropriate for CD4 Lesson 5 Modifying Group Analyses 41 ag E Bis Hie Ol fee E E i ee j Eee Ta a ee subsets In particular they 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 see 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 ees stained sample Fw L nr bill
73. most likely your table will only be applicable to one group of samples Go back to your Workspace and select the PBMC subsets group From the first sample only select the Lymphocytes and CD45 subpopu lations and the two frequency statistics under the CD45 subset shift click to highlight the four rows 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 Just as you can use the option Tuterial WS Chapter My Table Editor pe drag action on EB SSS P bee analysis trees between samples E rteiea Tab ED Lamphacshar you can also use EE Lyrreaeeyiecyc bei Froana A i Lympkeryier CPS Freg of Grencparer option drag for x Lymgptorytar t PS Freg of Parent transferring an entire tree to the Table Editor Lesson 6 Tables and Layouts Collating Data Output 51 Click on the Monocytes gate in the first sample and while holding down the option key drag it to the Table Editor window You will note that all five of the analyses are added in order to the table the Table Editor should now appear as shown below SSS Tutorial WS Chanter 06y Table Editor HH Ei tane ED kymphocy bes Fregi e3 Lumpo bee ODES Firag eny E Lyamphouy ter OF Preg of Crandperert E Lymphocy ber Soe St Freq of Parent EB roniva Freg iiey Lit Mon crbes Horo Frequecuns E ponscytesYMone Freg of Parent EH pionacates
74. nd Y axes to CD14 and CD45 50608 and change the graph type to Pseudo color Now click on the Annotate tab near the top of the window This shows a Ree Peer Oba asg DRE S Graph Tyee E Opiistri Keach Tyee Pndomeodar Pin a bleep Erie ay AC oars Ditech Ea High Beechrten Show ththers T 5 ee TH i ije since Pe FPapulsiim egiii Boation Tantops Tin ieatso f Chected Henr wont ary Tiik Pes oa H ear E in _ Cancel ser j Claes Gram Tals A Teys Boonies Hee E ra Hai Omera Clir iiei mere different set of options that control sd what appears in the graphic see right Unselect the checkbox Show Fr ioma mis anes cna Ciim elh ipt Annotation this will remove the mikar 1h isin 1 mE Cit ia Hir tr annotation text box below the maa Hore Daje wet Ee graphic Click on Set and the cme fl soe Lesson 7 Creating Simple Graphical Layouts 59 Layout Editor should appear as follows On screen it may appear that there are fewer events this is due to the reduced magnification in the window Verify this by selecting 100 in the magnification window When you copy or print all dots will show up Position Norigentaity or varticatiy Partially riail aiy L Aegn br Ialigis C testribute by C Mstribute bey i Lett to The Ciner tuntur Haii Erum Wwieh Height Cp Position by grii position Diba Bo rid ge ti firi
75. nd from the Lesson 4 Groups and Batch Analyses 35 if PEHE faboets oe APT ceil ka W i 2 irma w ensa Sanpie 01 for 7 famn T A reei Er E Ferg of Parr oO coer E Feng of Parvat Te popile bapatean T E rep ef Pret gt Psu cre Berpe DET iD Lampie iri Now we move on to the next set of analyses the CD4 and CD8 subsets Again we will use groups to speed up the process and ensure that identical gates are used for common gatings like lympho cytes While the CD4 and CD8 subset analyses used different stains we can still copy the lymphocyte gate since it is based only on For ward and Side scatter Click on the Lymphocyte gate from one of the samples e g the first one in the current group and drop it on top of the CD4 subset group The gate is 36 Workspace menu Since the cur rently highlighted row is a group FlowJo applies the com mand to all of the samples in the group i e the group serves as a substitute for each and every sample in its list After you perform the Recalculate command you should see that all of the samples have been com puted see example left We will also use the command Edit Workspace Columns to remove the reagents from the workspace to simplify our view You can choose to display or hide whatever attributes you prefer iS Tetorial WS fOhapter 4 T All Gamps k Mr M E E iyphidni Tr PF Teber TEY Epris heat suberte
76. ng only the events contained within the gate you just drew For now just click on the OK button You will note some changes to the Graph window The box in the upper right has the name of the currently selected gate and the fraction of events in the entire sample that fall into the subset In addition the Down arrow button is now active If you click on the Down arrow 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 left 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 Anything that can be done to a sample can also be done to a subpopulation You can double click on the Lymphocytes 18 Lesson 2 Gating and Statistics T SE B Pargle Bl fca_lypnpaocpies E subset to open its graph gate within g that subset etc Double click on this emin OET A population You will see the graph to the right This is the scatter distribution for the events falling within the lymphocyte gate Note that the number of events in this graph top left is 7003 which is the number of cells falling in the lympho cyte gate You wi
77. nss r rye ama Tedd at Frag ac Dae Ea e a Fe on graphs appear below Select the annotation text boxes under the three newest graphs and press the delete 0 SS nies weer in air o key to remove the annotations Move the graphs a little closer together see right Now you have created a graphical report that draws graphs from different tubes of the same patient Click on the I teration popup menu and select ID 1004 All four graphs and the statistic change and now are drawn from the fourth patient sample in the workspace You can select any patient sample 88 Lesson 9 Generating Complex Batch Analysis to view the graphs To view the tiled report just click on the batch generation button FlowJo now iterates only four times and generates four frames one for each unique value of Cells You should see a window such as here You can quickly generate single page reports one page per frame by select ing 1 Up and Scale To Page from the appropriate popup menus inside the Stack View window If you choose to print the Batch Output you will get four pages Finally as you learned before if you want to save the Layout Set tings select the Save Layout Settings item under the Layout menu You may wish to experiment some more by dragging in the folder for Experiment 2 on to the current workspace and regenerate these layouts They will be considerably more
78. ny sample s graph to verify that it Torem has the right lymphocyte gate If you then click on the group Exper iment 1 and choose Recalculate Samples all gates and statistics for all samples in this experiment will be recalculated After you have done this select the CD8 T subset group l an RH ea 0 ed E loth ch u S51 EH Ser OT ed El lyre l smyg sane Od er aD Leelee SD Hesseqee ST a ee Od er i Lar a Tope T Si oo r One final lesson before moving on to tabular and graphical presenta tions merging analysis trees Let s assume that you modify an exist ing 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 statis tic individually FlowJo gives you the ability to easily merge analysis trees that are very similar adding only the new or different analyses to the destination pre p aE p z p p os pre When you drag an entire analysis tree onto a population that already has parts of that tree then FlowJo offers four options 1 Cancel Do not change anything 2 Rename duplicates the tree Renames the analysis tree you are copying and attaches it to the subset as a copy 46 Lesson 5 Modifying Group Analyses 3 Replace the existing tree FlowJo deletes the old analysis tree and replaces it entirely with the new one 4 Retain
79. o FlowJo treestar com D et he FI FlowJo opens the Demo Workspace and shows you the window at right The organization of the Workspace window is more fully described in later chapters For now realize that the upper half of the window consists of Sample Groups which specify analyses gates amp statistics to be applied to appropriately stained samples and the lower half of the window is the list of all samples in the cur rently selected group All Samples always includes every sample in the Workspace Because this is a template workspace there are no samples in it There are several ways to load data into a workspace perhaps the easiest is to drag a folder of data files from the Tutorial Folder in the Finder and drop it on the Workspace sample list Click on Experiment 1 in the ao Me bd Fire tp Hele Teter a Drema aires pat eter Dj Hai aia A oo teeters 6 Introduction Finder you may have to move the Tutorial Folder so that it is not under neath the workspace and without releasing the mouse drag it over the lower half of the Workspace window 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 E ea aa ea i ER files collected on cytometers EA fe oN eo from any manufacturer The workspace now reflects the new samples and analyses right In this experiment each samp
80. off Show Descendent Gates You can create a History plot where FlowJo shows you not only the graph for a subset but the plots of all of parent populations as well E Tiria WI captor The Lava tier o Fee yin taper ios res e iad fees Pesha tals pi BS teat Cheeky r FlowJo gives you enormous flexibility in designing cus tomized layouts so that you can quickly and easily generate high quality publication and presentation graphics Lesson 7 Creating Simple Graphical Layouts 71 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 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 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 proces
81. ou wish just like a regular text box Deleting Grid Conk Damanis of miucini EC Peeters In this Grid the fourth column For is un maea anaa ritme vaih rimini used because none of the statistics here apply E rer et mete to a specific parameter like forward scatter lt r or fluorescence We can delete this column click on any cell in the fourth column and press the delete key FlowJo asks whether you wish to delete the contents of this cell or the row or column containing the cell Click on Delete Columns The next order of business is to reduce the font size in the table items so that they fit better Click on the top left cell in the Grid and shift click on the bottom right cell in the _ _ ae Grid this will select the entire f Il A 1 di th Lims Styles o Calar range of cells including thes en e ei cau two i e all cells in the table tent cron amp Double click on any cell with text mes se in it and you will see the win TetStyics ia dow to set attributes on multiple Ets ES items simultaneously As shown a s lal Ely te Gent cheng j here select a font size of 9 and i iinr EE a ai eee ee E 96 Lesson 10 Creating Finished Reports click on Set Next er resize the Grid to Se SS ee make it smaller Click on the lower right hand selection handle and drag it to make the table fit in the area of the Layout Editor You can resize indi vidual
82. ove If you wished to have a particular keyword value in a different font or color you would have to make a separate text box for it and format it accordingly The next job is to add graphics into the middle panel that show how each anti body staining panel was gated This is similar to some of the layouts you created in previous chapters First drag the ungated sample 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 momentarily However first drag in three other graphs into the Layout Editor as shown above select the T Cells graph from the second sample and the Lesson 10 Creating Finished Reports 93 two Lymphocytes gates from the CD4 amp CD8 staining panels Shift click these and drag them all at once onto the Layout Editor Your view will probably look something as shown here You will now change the attributes on these four graphs all at once to save time Select all four graphs you can use a Marquee Selection by clicking outside the bounds of all four and dragging to include them all or simply shift click all four Double click on the selection or select PENEAN LEEN Get I nfo from the Layout menu FlowJo shows you a display that lets you set attributes for all selected items at once As shown in this win wae ioe Bid it A kilid tu ge
83. over and over To generate one of these graphical reports you need to open the Layout Editor Window You can either select this from the Windows menu or click on the Layout icon in the Workspace window as shown above pen Soy 3 os 4 aT I z BSD Dr i All Samples FlowJo now shows you the Layout Editor window opening one of the four graphical layouts that have been designed for this workspace see graphic In the left hand portion of the Layout Editor window you will see a list of different layout templates Click on each one to see some examples of different layout formats Finally click on the Layout named Scatter Gates i y a Denies Yerke bait Beiter De One of the important first Ee mew a Perrier A fe steps in analyzing an ex periment is to make sure that all of the scatter gates are appro priate for each sample You will use this layout to verify the scatter gates for all 16 tubes collected in this experiment Change the magnification to 75 as shown here This gives you a better view of the Forward vs z Side scatter dot plot If you wanted you could now cycle through every plot by clicking successively on the Next or Previous sample arrows just below the Help button However you can use FlowJo s Batch analysis to automate this process Click on the Batch Generation Button it looks like a stack of papers right below the Help button 8 In
84. peri ment 2 there are 14 The stain combinations used are as follows FITC CD14 CD8 CD4 CD62L CD45RA CD4 CD62L CD45RA CD8 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 les CO nora paee I IE about subsets AR eaaa Once you have launched FlowJo select New from the File menu You will be shown a window similar to that shown to the right Note the Help button near the top right Most windows in FlowJo have a Help button When you click on Help FlowJo attempts to launch a web browser to get the on line version of Help or you can Lesson 1 Workspaces and Basic Data Display Il press the Help key on your keyboard at any time The browser chosen can be set using InternetConfig the default is Netscape Navigator The help for FlowJo is HTML based when you ask for help you will auto matically be shown information about the active window From 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 Help under the Apple menu 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
85. pl ry Any graphic item can be made into an overlay by dragging any other subset and dropping it onto the overlay You can overlay different subsets from the same sample or overlay plots from different samples For now select raph Axs Ha aE Coa Tiot Fleer Goel 4 cee Options Beaph Tapes Det PRE Fy i e A TA Frakak Urrea Limani hibernis thes Eee rE T ae RT the Monocytes subset from the ea ms hoo be treme j same sample and drop it on the graphic the graphic will highlight to let you know that you can drop it to create an overlay You should 66 Lesson 7 Creating Simple Graphical Layouts now see the multi color dot plot shown at the bottom of this page FlowJo is drawing the two dot plots one for each subset in the same graph In addition it automati cally creates a Legend for the overlay shown to the right of the graphic 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 order in which the dots are drawn by clicking on any item and moving it up or down in the list Finally you can delete an item from the overlay by holding down the option key while clicking on one of the subsets the cursor will temporarily appear to be a trash can when this happens You can over lay an almost unlimited number of different subsets on the same graph SSS iri ws e a a Note you can
86. r Aiya apl ihi y allia oF ames e For now choose to Align by Top and click on Set The Layout Editor will reflect your changes Sometimes adjacent graphs have exactly the same Y axis or X axis 60 liaii 71 apii DE Tadei FlowJo provides many features of draw ing programs such as the ability to align multiple objects To align the two graphs select them both shift click on the graphics or use a marquee selection tool by clicking and dragging to encom pass both graphs and then choose Alignment K from the Layout menu FlowJo brings up the Align Ob jects window shown left You may choose to Align or Distribute objects in either or both dimensions j LL ag 7 Lesson 7 Creating Simple Graphical Layouts FlowJo lets you remove the axis labels and align the objects to be adjacent to each other easily creating compressed graphical presentations suitable for publication Double click on the right most graphic and under the Annotate options choose to hide the Y axis see right Now select the two graphs and choose Alignment again Now you wish to align them by the top edges and the align them adjacent to each other Align by Width In addition you can ask FlowJo that it aligns graph items by the position of their axes rather than by the position of enclosing rectangle check the Align graph items box The Align Objects window sho
87. r confirmation window shown below Confirm action FlowJo is warn ing you that the Some gates you are dragging are based on stains that are CD4 gate was notin a destination sample Do you want to apply these drawn on PE anh sisi CD8 vs Cy5PE CD4 and may Oo Always use same answer not be applicable to the sample stained with PE CD16 and Cy5PE CD45 In fact in this case the gate is not appropriate You can if you wish choose to copy it anyway and then the gate would be applied to the PE and Cy5PE channels irrespec tive of the stain 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 properly 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 Note that there is an option in the Preferences to have FlowJo periodi cally ask you to save the workspace and by clicking in the Use Same Answer checkbox the first time FlowJo asks you to save the workspace it will then automatically save the workspace every few minutes Lesson 3 Copying Analyses to Other Samples 31 Lesson 4 Groups and Batch Analyses In this lesson you will learn how to take advant
88. reen and the style to italic When you are finished click Done Your Workspace window after you click on the T Cell Analysis group should look similar to the one shown here AMHER Each group is displayed in the upper group list in the color and style you have chosen 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 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 differ ent groups Finish by selecting the T cell analysis group 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 Hold down the option key while dragging 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 happened First the analysis tree has been added to the group row as if it were a sample Second the analy 34 Lesson 4 Groups and Batch Analyses ses have been added to every sample in the sample list Third all of these analyses now appear in blue and italic which is the same color and style that you defined for this group You can
89. requency fel Sten Count Sine 2g astroh HTAL Output Bejn okapi pa A a bre Qor Omt Om memm aw Header He Fike Spectied Crome EFS sie 12 2 J gustitination Left Pooler Me Fiki Spc ified oars chee J Siyi Pran Diras Border Ir piak ahr U Bae ra fel lor caper ore A e ae mero fer j en annb wor deer i oaio the onsale l Cancel Save 80 Lesson 8 Creating Batch Graphical Reports 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 well as graphical reports that draw graphs from multiple different tubes for each frame This lesson builds on the Workspace 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 Layout View should appear as below Tutorial WS Chapter 9 Layout Editor Complex Repo a Scatter Analy Overlay Layout 0 1 td 931115 B01 Samplke 01 fes Event Count 10000 E E T E D Lesson 9 Generating Complex Batch Analysis 81 In the Workspace click on the Freq of Parent statistic under the CD45 gate right Drag this statistic into the Layout Editor holding the mouse button down as you move the statisti
90. rows and col umns by clicking on any dividing line and dragging Resize the columns so that the full text is shown in each cell see top right 5t 7 La tabr da tinier Finally while the Grid is still selected if z salen oT choose Get Grid I nfo from the Layout Reape Menu Here you can select specific at tra pae e sates tributes about the Grid Set the Inner fon etna patie whi Dashing to Short Dash the fill color toa an light Yellow and the Pen color to dark blue Hiesseatisutrussmirid ens items wu Click on Set when you are done For more information about other Grid at ao ianen J to tributes you can click on the Help button If you increase the Hea i magnification on the Layout Editor you should see your table similar to the one show here Now it s time to create the other Grid In this Grid you will Lesson 10 Creating Finished Reports 97 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 To create a blank Grid click on the Grid tool it is the one right above the vertical scroll bar at the edge of the Layout Window Click and drag a rectangle that occupies the
91. rry EJF re cree pease rears ii Lap ae Rk damal elie Ligand Mares TEI thew damri L fmn eas fem Gji eae Uray A irmi Djia oe i ms ay ka oe Fes nh Ba Hi O rri E at Fe k Eo Bpi ie pa a Pop oe Aia EEE rag a JE 1 hee dow change several attributes First check the Inverted check box in the Graph Type op tions Next click on Show Annotation Box twice a check mark means that the attribute is On a blank box means the attribute is Off and a dash means that some items have the attribute on others have it off and the setting should not be changed for any of them Also check the boxes for Show Gates and Show Gate and type in values of 40 in the two scaling boxes near the bottom When you are done click on the Set button All four items are changed as you requested 94 Lesson 10 Creating Finished Reports Now move the graphs into different locations perhaps as shown in the example on the right here the lym phocyte amp monocyte gates are shown first then the CD4 amp CD8 gates on the CD3 subset and on the right the CD4 amp CD8 gates Note that each graph is derived froma 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 Finally you can create some text boxes to annotate the graphs In this example the n
92. s a unique value for each different Patient sample In the demonstration data there heratios Options Ering Te Frat iF Pat he Li i eee Ao 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 you may later wish to add that data to play with a larger data set Fut bby Tahte Jorin ih zaam Qut We will now tell FlowJo that it should iterate over patient samples To do this select Iteration Options from the Layout Menu see left Lesson 9 Generating Complex Batch Analysis 85 FlowJo now shows you a Lay tteration Options window where you can Sf Allow Batch Operation _ 1 i Etoh Pepet iega HE b berrik ra Al ngis i poan ie specify which FCS key ord ere tspererated kr each unis vane of the sertie Herlin kryera should be used as the con F sta wiri bo genet oea Fam Por aray tuber ihan iieri Tarp h 3 Beran i troller for Iteration By aa e Choose the bryenmd aboar rakes ber dl default for new Layouts ne rat ic a Hoyos ole beysrord ta certenl Herston tH this is set to Sample mean af dene tale cbs rant Ki ing that each tube in the ee er Ept Gamer prahe shake gkh tih irag Workspace 1S a unique argh aka igi abt de ibm iteration value AS shown ina Cor irnia ba beye ed k te cama Set ti i A ee Hamp By in the figure select Cells Chew tha kee hee vee Re i xili migel entity an th
93. s where it needs to be Lesson 5 Modifying Group Analyses 47 E Euficrial Wg Chapter T nae E iry eiie Te adn SO aren Or tee Ta Ly ergo rg bo ise eat attached FlowJo now brings up the dialog shown below Here you can choose between the four options listed above If you choose Rename then FlowJo will rename the Lymphocyte analysis that you are dragging and call it Lymphocyte 1 FlowJo has to rename it because two subsets with the same name cannot coexist Alternatively you could select Retain This instructs FlowJo to add any analy ses that are different between the tree you are dragging and the existing tree but not to modify the existing tree in any way Thus had you modified any of the gates in C08 like the lymphocyte gate so that they were different from the ones in B08 FlowJo would preserve those changes However the statistics would still be added in the appropriate places Or finally you can select Replace This option instructs FlowJo to delete the existing Lymphocyte subset tree and replace it with the tree you are dragging Any sample specific modifi cations that you had made for the analysis tree in C08 would be 48 Duplicate Gate hesoher Horw houihi gates wiih the same names we handien Theres are they diferent schonr Wiat mighi make ere here Toa p baii watt bo tee Pople Di Tet i aa e a pr ar ark Replace The new amel gsi gales should replace EXIST ng orts o
94. sing 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 displayed and selected in the Iteration Popup menu which is just the below the Help button in the Layout Editor Whenever you create a new Layout the current Iteration state for that view is set to Off When Iteration is off then 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 72 Lesson 8 Creating Batch Graphical Reports If you click on the iteration popup menu you will see a display such as that below O Tutorial WS Chapter 8 Layout Editor E E Ren om ea Mame Sather Analysts tee Praceneagers ae w Sample j oT FEI E BO pompk ml TeF S21 5 H Sanple Dri tee 221 iTO Samph Di tes FCL 3 80 bomp tes 13511 3 20 mmp Ne Ter 37i 1 15 0 Geel OS to
95. t FlowJo instantly updates the graph of the subset that window will now look like the one below right 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 you make minor modifications to a gate like clicking and dragging a single vertex Move the gate back to the lymphocyte population so that the gate accurately reflects lymphocyte cells Change the graph for the lymphocyte population click on the Down arrow to get to the Lymphocyte Graph window or simply select the corresponding Graph win dow so that it displays a histogram of CD45 Select Histogram using the Y axis pop up or use the Graph Tools palette to define the graph type 20 BY giii argie dikni E ESE i El FISE y 10000 21 Se Lpa ia 100 7 eo el MimED d Lesson 2 Gating and Statistics The graph should now appear as shown on the left If you wish you can change the vertical scaling on univariate plots through the controls in the Graph Tools palette 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 Click on one side of the peak and drag to the other side When you let go of
96. t FlowJo uses the CD4 T CD8 T and ae ena dTi 100 aia bE a oeutte Megat on 7 eae Lomphergies T Cele Cy BP Coa Lesson 3 Copying Analyses to Other Samples 27 Double Negative T D SSS eral aer ss BS j i zje 2 3 subpopulations are sib shares i i k lings and are children of ol wero the T cells subpopula EC a r E j tion grand children of ET oe TE 100 Te the Lymphocytes sub population etc Remem ber that the frequencies and cell counts shown to the right are with respect to the entire ungated sample The Workspace window above reflects this hierarchy Phe ee ee PI ELTS baraka EI ea 8110 00 Barghe ES foe To calculate the relative representation of these three subsets within T cells add the Freq of Parent statistic to each subset You may also add it to the first subset CD4 T using 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 below You could also select all three subsets shift click them and add the statistic to all at once from the n ira 5 i es 7 Statistics window OMAA AE l i an Samples Click on the triangle next Hy to the Lymphocytes gate to collapse the view A second
97. tatistic node present there would also be a blank entry The column heads show you the name of the subset s parent gates ancestry the name of the subset the statistic and the parameter on which the statistic is calculated For 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 Optionally you can show the Mean and Standard Deviation of the statistics at the bottom of each column If they do not appear check the Preferences window under Tables amp Layouts and check the box marked Show Summary Stats 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 Lesson 6 Tables and Layouts Collating Data Output 53 Workbook ae i is ET i 65 ane oe ko at To export the table click on one of the four buttons near the top left The first button will save the table to disk as tab amp carriage return delimited text data The second button copies the table to the clipboard and you can then paste it into a spreadsheet application for ex
98. tch Output FlowJo will start with the first iteration value and generate a frame for that particular sample FlowJo then continues to generate anew Frame for each successive Iteration Value until it has exhausted the current group These Frames are now shown to you in the Tiled Report window such as that shown here Batch Layouts can be displayed in four different modes 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 of specific graphs or by removing graphs that are not interesting The second mode is a Tiled Report in which you get a new window containing each of the tiles laid out on a special page grid The third mode of reporting is a Web Report where FlowJo creates a folder containing each frame as a separate graphic and creates an index html file to easily view the graphics from the World Wide Web The final form of reporting is a Quicktime Movie where each set of graphs becomes one frame of an animation You can play the movie in FlowJo or any movie viewer and cycle through all of the graphics All of the different views generated in these batch reports support print ing saving to disk copying to the clipboard and creating Web pages 76 Lesson 8 Creating Batch Graphical Reports Back to the Tiled Report view There
99. tely analyze and generate custom graphics and tabular reports in a matter of just minutes for an entire experiment Dania Workspace Layout Editar E ered Hirs ether ma This is the true power of FlowJo Experiment Based Analysis Introduction Lesson 1 Workspaces and Basic Data Display This lesson includes an introduction to FlowJo You will learn how to start the program along with the basic concepts of the Workspace how to load data and how to view some graphs 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 and 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
100. the statistic value you want FlowJo won t let you edit the command unless you check the box Edit FJ ML Directly 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 this i bert rade for Kirrwerd Gulect T Cheat FAA Drect Teddi g matitie drap attrac tie Workapaor and drop Ms the INF Heip Update Layout cancel ox Edit Layout Tet pH You can click on the Lymphoeytes ara Statiatic Update button to see peert 45 7 Lymphoeytes CD45 F reg af Parents of the the changes to the Layout View or click on OK to accept changes and go on You can leave this window open in order to drag other statistics into the text See Chapter 10 Note that you can also select keyword values to add to text boxes the keyword values are data encoded in the file itself You can create additional text boxes with more statistics by dragging them from the workspace You can also add ts insert wue for keyeard Sete E Edit Feb Directly To ede a Pe Ge eg fete She Werks aaa Af in he tee Help I Update Layout Cancel K Fe Lesson 9 Generating Complex Batch Analysis 83 multiple statistics to one text box either drag another statistic into an existing text box or
101. troduction From here you can choose from a variety of report formats Leave the option set to New Layout to see a report like the one below Or play around with the different options the Tiled Report to see a matrix of all your plots Web Report to view the output in a browser or Movie to play each successive sample in frames of an animation In Chapters 8 and 9 you will learn how to arrange the graphics in the Tiled Frames view to be printed in exactly 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 Mate Batch Raparii Datpri BCR parc a bra ety aa kT big differed wan et poetry the ata D Bow Lagu 28h Bapt QP Tiled Beport Gf Herria Lerat Nana t giar ath Perala The reper bpr ad by for sah rgi ni a Te ee ery Dee Warkcoae s Correa Grop ee ee Pargis Sein Saree 2 izemiry Flaws the ew in fa i iiia m Clann eden drs piran O tore Tarm Can ira ered Ha Origa layami CI te Stee Th a aries ia Ey br aren ET Cj Experiment 2 This is data for 14 more individuals and is therefore much more extensive than Experiment 1 Drag this folder onto the Workspace like you did for Experiment 1 and H generate the batch out puts as before EI abating rekr EY Faisan E rey aargh E Sarrar Osira Elim ibh ilir ahi This example should convince you that with a well designed Workspace you can comple
102. uld look as shown in the middle figure at right Now the Layout Editor shows the two graphic items 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 ipm BES Beko eo Pepetabim Ustad CLayaut Rem Definition OT ete Perit Mee A ee T A hid deeds fore ee raa EA iie Dak bere m Bf cas Tier as 0 akaw Laqeed iline tyies ratr Dilber DeHings Hels bows Tka Ami Eres Fea irii Aras Clr Plath Oike irii Us a pitsi u itj Ibhi rai fix ug Hri TA Mis Align Shiacts Positien horteontally or vertically We kite Ber oie diem 4 Align by lA Alg iry Ci strie oy DSi by ILER lap Diemer lt HRight amp rii D Positiss try grid porises isiriiiae to Grid Aligi Eh Orin by side giving you complete a flexibility in displaying graphs HF The layout will now appear as shown on the bottom right FlowJo s Layout Editor pro vides a few simple drawing Lesson 7 Creating Simple Graphical Layouts 61 tools to elaborate your graphical reports These tools can be selected by clicking on one of the tools near the upper right edge of the window as shown here You can choose a Line tool to draw lines and arrows a Rectangle tool to draw filled or unfilled rectangles a Text tool to create text objects or a Grid tool to create grids
103. v3 reference 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 In addition there is a web page for FlowJo FAQs Frequently Asked Questions 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 wish to subscribe to a FlowJo EMail discussion group where you can post questions answers or tips For more infor mation direct your browser to www flowjo com Finally we are pleased to be able to frequently update FlowJo to pro vide new features amp analysis capabilities Therefore it is possible that the graphics shown 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 Mitroducti n enner Seed ear cua a a id teate chap aaah emcee aceaeoee 5 Lesson 1 Workspaces and Basic Data Display ccccccssssseeeees 10 Lesson 2 Gating and Statistics ssseeseeseseeseseseseereesessesessesssresessessese 17 Lesson 3 Copying Analyses to Other Samples ccceeeecseeeee 26 Lesson 4 Groups and Batch Analyses cccccccssesseseseeeteseeeessseseneeees 32 Lesson 5 Modifying Group Analyses s s sssssesessesssresessessesissessssese
104. while it computes statistics You can even have it start many different tables computing simultaneously then go back and analyze more data FlowJo schedules the calculations so efficiently that it s actually faster if you create multiple tables amp layouts simultaneously 56 Lesson 6 Tables and Layouts Collating Data Output Lesson 7 Creating Simple Graphical Layouts In this chapter you will learn the fundamentals of the Layout Editor 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 Tuto rial WS Chapter 7 To open the Layout Editor either select Layout Editor from the Windows menu press 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 Editor window the left portion of the window is a list of all of the layouts you have created for this workspace You can have as many as
105. you can select the line weights Plain Bold or Heavy if the histogram should be filled Solid or if it should be drawn as both a line as well as filled Select Solid Then click on the control next to the Blue monocytes color box and select Bold This will edit the line weight and the fill color of the two lines in the graph Your Layout should now show the overlay histo gram as shown in the bottom graphic Note that if you change the order in which the subsets are drawn you can have a substantial effect on the output To see this click on Lymphocytes and drag it up so that it is first in the list eC ie te a an e a EEG ee el To ama ba r E Tetona Sa aper Ti Lavat Caine r rad kaa A papune i Ee D 70 Lesson 7 Creating Simple Graphical Layouts To finish up switch back to the Scatter Analysis Layout You will note that the monocyte gate is still incorrect To correct the layout open the 931115 B01 sample again and move the monocyte gate to the correct position This will cause FlowJo to automatically recalculate the affected population and all child populations which are affected indirectly because the gate determines all child populations The picture to the right reflects the fact that the gate moved There are many options to changing how your graphs appear You can choose to have FlowJo show gates drawn on a population as it is here or leave them
106. you wish you will probably want to create different layouts for different purposes 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 lay out by clicking in the Name field near the top of the window and typing a new name To add a graphic to the Layout click on any subset in the workspace 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 Lesson 7 Creating Simple Graphical Layouts 57 contains some pertinent information Most of these options have a default value in the Layout Preferences so that you can tailor the information added to your graphs in the Layout Editor Any graphic item can be resized or moved just by clicking and dragging To resize click and drag on one of the four handles at z each corner If you hold T down the shift key while resizing the graphic main a tains its original aspect ki ie Sa bigs ratio You can also change Pliage the magnification of the view by clicking on the Scale popup menu near the bottom of the window see example

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