Protocol to prepare flow cytometer for acquisition of samples
Contents
1. acquisition box will appear telling you that this has been done successfully At this point you need to either open or design an acquisition layout To set up a new one select under the file pull down menu new A blank screen appears Now dot plots and histograms need to be added as you wish The only sort of mandatory screen you must have is the light scatter screen which plots forward scatter versus side scatter To get this screen click on the palette for dot plot and select acquisition in the inspector window that appears select no gate and single color Click on the polygon creation tool and create R1 in the plot somewhere i e where you expect your cells to be where lymphocytes normally are etc Note to view details of a plot in the inspector the plot must be selected so that there are handles on the corners by clicking on the white border of the plot Now click on the dot plot icon or the histogram icon to select other acquisition parameters based on fluorescence As in the previous one select acquisition but this time select G1 gate The reason you select the G1 gate is because you will eventually move the R1 in the FSC SSC plot onto your cells of interest By selecting G1 it means your fluorescence will be reflective of the R1 population and not contaminating cells be they of a different lineage dead apoptotic etc If you have a layout saved go under file open and select the a2. gate around all white cells and do your analysis the backgrounds are different for each population and this might make analysis tough or give false results Make subsequent dot plots or histograms the same way plot source analysis select file to match the file used in the first plot choose your x and y parameters they will not be FSC SSC anymore but will now be the fluorescence parameters gate G1 R1 because those are the cells you are interested in Region gate viewing deletion Go to under the gates menu Choose region list Note that R1 is there because you have just drawn G1 R1 on the FSC SSC plot All gates you make are shown here If you make a mistake and either mistakenly draw a gate or make one that is totally oversized etc then come to this location highlight the at the side of the gate and hit delete on the keyboard to delete it Fluorescence parameter choice Assume you made a mistake in choosing the fluorescence parameters on the plot To change fluorescence parameters once you have closed the new dot plot histogram creation page just click on the parameter on the dot plot histogram on the screen at that time a box will appear with all the parameters and you can scroll down and change it Region gate viewing deletion 2 Make R2 somewhere on the second fluorescence dot plot Now go under the gates menu choose regions and now you should see both R1 and R2 listed under here bec
3. may be billed 1 hour of usage time Please update your reservations as soon as you know they are different from the scheduled time Late deletions or no shows may be billed 1 hour of usage time General Guidelines 50 per hour for user run analysis tracked by how long user is logged onto the computer Use the dedicated analysis workstations for free instead of getting charged for using the instrument computers for analysis Wear gloves when using instruments No gloves on analysis stations NEW WASTE PROTOCOL All Flow waste must be disposed of as hazardous waste and is collected in a plastic drum that is kept near the calibur in 5228 On the Calibur there will be an empty cube near the fluidics cart When the active cube is full switch it to the empty one and the full one will be transferred to the drum If I am not around and both are full please wear a lab coat and empty them as follows On top of the waste drum is a bucket with a funnel and a spigot Attach the spigot to the waste cube and make sure it is in the off position handle to the right Remove cap to drum and position cube on top of drum so that spigot is directly over the opening Open the spigot and allow the waste to flow into the drum When nearly empty close spigot use the funnel to transfer the waste to the drum and replace the cap on the drum when you are done Also remember to add bleach to the empty waste container Using the FACSCalibur Tutori
4. Do not let the tube run dry as it will make your data file a very poor one and screw up your results too and even if it s dry it will keep collecting air as events and not stop running If this happens tell someone who works in the flow facility When you are done collecting your samples close down CellQuest go under file and select quit Also close all boxes on the screen Bleach the cytometer for 5 minutes and then run water for 5 minutes change tanks etc Clean the cytometer as instructed on the back of the user guide binder Finally transfer your data files to a remote site the server then delete them from the temporary folder on the hard drive Nothing can stay on the hard drive We delete it from time to time without notice it is done every 2 weeks and if your data are left they will be deleted without notice How in what order to run tubes on the cytometer assuming a 4 color experiment 1 Unstained cells OR cells stained with negative control antibodies This tube is used for 2 things First adjust forward and side scatter parameters by adjusting the FSC and SSC controls You are aiming to have all cells of interest on the screen Also you aim to have them in the right spot which is arbitrary What I mean is this there s no difference in your data if you put say lymphocyte scatter at the center of the plot rather than in the lover left hand corner of the plot However people are not used t
5. FACSCalibur User Guide Ragon Institute of MGH MIT and Harvard at Massachusetts General Hopsital Visit our facility website http www2 massgeneral org aids flow cytometry html It contains information about our instruments links to calendars and flow resources and many of the documents that I hand out as well as a flow cytometry syllabus of resources to expand your flow knowledge Index FACSCalibur Optical Bench Configuration Pri 530 30 ssc 488 10 Er 585 42 90 10 Beam Splitter Dral 661146 DM 5605P M 640L 670LP Half Mirror Fluorescence Collection Lens PFL Beam Combiner Red Diode Laser 635 nm Focusing Lens FSC Diode 488 nm Blue Laser 488 10 FITC GFP CFSE Alexa 488 530 30 PE PI Pyronin Y DsRed Alexa 546 585 42 PE Cy5 PerCP PerCP Cy5 5 PE Cy5 5 670LP 488 10 APC Alexa 647 Cy5 661 16 default parameter dimension is H DDM allows measurement of A and or W of one parameter Calibur user guide To add Cellquest pro to your program dock at the bottom of the screen drag the icon from the BD Applications gt Cellquest Pro folder onto the dock Do NOT drag the icon to your desktop which will physically move the entire program and prevent anyone else from using it Creating an Account To become a user Visit www2 massgeneral org aids flow_cytometry html and go through the introductory material on the training page attend the introd
6. ITC fluorescence out of FL2 The only real sure fire way to do compensation is electronically but that can not be done on the FACSCalibur currently so you have to do it by sight or by quadrant analysis Note you only have the option of correcting FL1 vs FL2 If you are running FITC and FL3 PerCP etc and NOT PE you may see overlap into FL3 with your FL1 label This would normally be corrected by adjusting your PE compensation In this case even though you do not have FL2 you could adjust FL3 FL2 to correct overlap of FL1 into FL3 Please see me if you need help with this 3 Cells Stained with PE ONLY See info about low percentage staining in FITC above it applies to all your stains PE can overlap in two directions with FITC and with Percp Tricolor PeCy5 PeCy5 5 7AAD these are all stains used in the FL3 channel In this case you need to worry about compensating out of the 2 channels FL1 FL2 and FL3 FL2 Remember though if you used PE and let s say FITC too but did not use anything in the FL3 channel then don t worry about compensating out of the FL3 channel you will never look at this channel because it has no stain and therefore no data 4 Cells stained with Percp Tricolor PeCy5 PeCy5 5 only Percp and the other ones in this channel can overlap in two directions with PE and with APC In this case you need to worry about compensating out of the 2 channels FL2_ FL3 and FL4_ FL3 O
7. R5 Type in for this scenario R1 AND R2 Two things of note the equals sign is there and all typing in capitals it MUST be in capitals The most common are AND AND NOT OR However any other boolean combination will work A note of caution if you make G5 be R1 and R2 and end up making more regions on the screen when you make RS you need to go back into the gates menu and make G5 R5 or else it will still be GS R1 AND R2 and you results will be completely wrong Changing gate colors or histogram colors If you want to change the color of a gate that is what color the cells appear on the screen that fall from a particular gate go to under gates choose gate list All other gate combinations will appear as well as color beside each Click the color and a palette of colors will appear Choose the one you want and close To change histogram colors go into plots choose format histogram and the menu will appear On the left hand side about 1 2 way down will be a box the default color is purple Click it Choose the color you want or no fill to just get a line with no color inside You can also choose the color of the line if doing no fill click on the line under the color box and choose the line color you want Statistics Region stats shows you the percentage of cells in each region on the plot both gated and ungated total So if you are looking at PBMC and
8. RONLY suggested you at least put the date of the expt here as well as any other info you may want although you of course can name the file whatever you like Be sure the file counter is set at 1 Click ok Under acquire click counters This lets you watch the number of cells acquiring in your file Now you are ready to begin acquiring your sample Change the cytometer from standby to run and hit the high flow rate button Make sure your acquisition control is in the set up mode by making sure there is an X in the box that says set up Put your sample on and click acquire First adjust FSC SSC parameters to make the cells appropriate in terms of light scatter Make sure that your cells of interest are in R1 Unless you know what you are doing set the threshold at FSC primary between 18 52 can t be explained here in this space If you DON T know how to do this ask you should not be using the machine by yourself in the first place Once all your light scatter fluorescence parameters and compensations are appropriate hit abort on the acquisition menu Take the machine off set up mode and hit acquire The machine will collect the number of events you told it to before However keep an eye on the tube if it seems you are not going to get the desired number of events then when the tube gets low on the acquisition box hit pause then save and your data will be saved to the file
9. Write Disk If you choose write session you can later burn more files to this disk in an additional session if there is space available e Verify disk will check to see the data was copied correctly e Remove disk label disk delete folder from desktop if you no longer need the files on the computer Analysis station Inserting a blank disk should cause a cd icon to appear on the desktop Simply drag and drop folders and files you wish to burn onto the cd icon and when all desired files have been added drag cd to the trash which will change to a burn symbol You can only write to a cd once using this method
10. affect other files of yours or other people s files KS statistic This stands for Kolmogrov Smirnov statistic and provides a D value which is capable of detecting very small but significant shifts in fluorescence based on the mean fluorescence intensity of the peaks The only time I have ever seen this used is to analyze the presence of the P glycoprotein antigen on clinical samples where the levels are significant but VERY low This statistic needs to be validated on controls usually and as a result do not use it unless you know in advance this is the statistic you need To present this statistic for any reason to denote differences in staining on 99 99 of staining experiments in just wrong To annotate plots add text Go to plots select annotate from here you can have displayed the file name patient ID or any other information you enter into the file while you were collecting it or you can go to the palette and click on the icon A which is the text tool and type your text Rearranging what is on the screen and how you see it To get multiple pages Go under file select document size Pages will be displayed click and blacken the of pages you want vertically or horizontally To size reduce what is on the page which allows more things to fit on the page Go under file Choose page setup Change the 100 to 90 80 70 I think 60 is the lowest you can go To analyze a se
11. als for use of the Calibur and Cellquest Pro for acquisition and analysis can be found on the Mac analysis station in room 5233 FACSAnalysis HD gt BD Applications gt Cellquest Pro Folder gt CQPro Acq Tutorial pdf or CQPro Analysis Tutorial pdf or online at www bdbiosciences com see our Training page for link MECHANICS OF MACHINE USE Calibur __ Turn on the cytometer first and then computer If computer is on and cytometer is off turn computer off and then start up __ Sheath and waste are regulated by the fluidics pump under the bench If the alarm sounds press the alarm button on the left side to silence and see which light is on Full sheath cubes are to the left empty waste tank should be in the cabinet to the right has hazardous waste label on it Change the appropriate tank and then press the restart button on the right side of the console Leave full waste tanks in the cabinet I will empty later Empty sheath cube goes at the end of the bay for trash pickup __Label all tubes Do not overfill no more than 2 3 full _ Push tube all the way up onto sample injection port move tube support arm to center position Black knob should NOT touch tube something of a misnomer doesn t actually support the sample tube __Don t let a tube run dry don t leave a sample tube on while tube support arm is to the side aspirates sample to waste __Filter samples especially if there might be large clumps of cells __ Drop
12. ause now you have made both R1 and R2 on the screen Now close the menu Go to the R2 gate click it and hit delete Go back to under gates regions and notice R2 is still there the only way to permanently delete it from the record is by deleting it under this menu If you drew ANOTHER gate at this point it would appear as R3 because R2 was never deleted from the menu Once you accidentally delete R2 from the screen or any region from the screen for that matter you can bring it back by selecting it in the region list copy in the Edit pulldown menu select the dot plot and select paste Gate combinations Go under the gates menu choose gate list G1 lines up with R1 G2 lines up with R2 etc This means that if you choose G1 for a gate then it will display all cells in R1 If you choose G2 it will display all cells in R2 Let s assume however that you want to look at a combination of gates that is all cells that fall into RI AND R2 all cells that fall in R1 but not in R2 all cells that fall in R1 OR R2 you see the logic here I m sure In order to do this under the gate menu you change the R column So if you want to look at all cells in R1 and R2 then go to a higher G anything G3 or above I usually go to G5 or above in case you make any more gates although this is not necessary Leave G5 alone In the corresponding column on the right highlight and delete
13. cquisition screen Now that the screen is set up how you want it work left to right in the pull down menu Under cytometer select detectors amps threshold and compensation Move them to the side of your acquisition screen If you have previous settings click instrument settings and select them be sure you select the file hit set then hit done watch the screen to be sure they have changed If you need new settings change your FSC and SSC to linear unless collecting dendritic cells and all the FL1 FL2 etc the fluorescence detectors to log around 500 on each PMT This is a good starting place if you are making new settings Now go over to under acquire and select acquisition and storage There will be a highlighted area of the screen it is a number this is the number of events you wish to collect Standard is anywhere from 10 000 to 200 000 depending on what expt you are doing Type in the number Then on the bottom of this screen check the saved parameters Make sure all the boxes have a check in them if not click them with the mouse so they do this prevents your data not being saved It is suggested you do not change anything lese on this screen unless you absolutely know what you are doing Now go under acquire and select parameter description Click folder and select to what folder you want to save it to Then under file name the file it is ST
14. f note APC and Percp are excited by different laser lines 633 488 respectively so the compensation is actually inter laser compensation However it does not change how you will do the compensation but it is why the time delay on the instrument is so important This gets really messed up if the machine is either a let run overnight or b let run dry for anything over10 minutes or so 5 Cells stained with APC APC overlaps with the FL3 channel so you have to adjust FL3 FL4 only 6 Your sample If you followed steps 1 5 you should not have to touch anything just put your sample on and acquire it should look perfect Flow Cytometry Analysis using CellQuest Open CellQuest Open an analysis template or make new one with dot plots histograms as you wish To make new analysis screen start with a light scatter screen Go to dot plot menu For plot source select analysis Click select file and then find the file you want to analyze X fsc or ssc Y fsc or ssc Gate no gate Show 100 of dots or if there are many events in the file eg an ICS or stem cell file then choose a lesser percentage than 100 so you can discriminate between populations of interest Using the polygon tool on the palette draw a gate region around the cells of interest for most people it is usually lymphocytes but if you want to look at other cells then you do this the same way In general with a few exceptions do NOT draw a
15. he following To purge air with tube on sample port press prime as soon as you see bubbles in tube press standby Depressurize by flipping black switch loosen cap to sheath and press prime run wait 3 sec prime run wait repeat 12 times then press standby pressurize system check sample voltage while run on high Then Discard the following files HD gt sys folder gt prefs gt BD prefs gt calib file HD gt Sys folder gt prefs gt BD Prefs gt FACSCompprefs HD gt BDFiles gt Calib file empty trash restart computer rerun FACSComp FACSCOMP Target Values Lot lt 69000 gt 69000 SSC 181 175 FLI1 74 63 FL2 70 58 FL3 65 54 FL4 SEE APC BEADS INSERT FOR VALUE FL1 FL2 5 24 FL2 FL1 14 9 FL3 FL2 24 21 FL3 FL4 65 65 FL4 FL3 41 41 Troubleshooting Rebuild desktop You can rebuild the desktop It s recommended to do so once a month Hold Alt and Apple keys down while clicking on Restart Continue to hold Alt amp Apple keys down until screen ask if you want to Rebuild Say yes Hope this helps No events during acquire restart computer verify cells in tube verify backflow of sheath To burn data to a cd Mac You can only use CD RW CD R DVD RW or DVD R on the Macs not Insert a blank cd Default should open the program Toast and the process is pretty self explainatory but if it does not open use the search tool in the top right of the window to find the application and open it to b
16. let containment system outer droplet containment sleeve tube support arm turns on vacuum and puts machine in a standby mode run button turns orange __ full standby button lasers power reduced stops sheath fluid flow to conserve sheath Use only when done while running samples leave instrument in run __PRIME flushes the flow cell refills with sheath clears air bubbles which may occur when you let sample tube run dry or if sheath tank runs dry and air is pumped through the system May eliminate noise Always remove tube before priming __Cellquest Pro Acquire gt connect to cytometer Open layout acq Settings instr Settings __ Proper shutdown procedure 2 3 full tube of bleach set tube support lever to side set to run on high for 1 minute about 1 ml then return lever to center position to run bleach through sample injection tube for 5 minutes Repeat with water leave water tube on Sample Injection Port with 1 cm of water in bottom press standby Do not leave too much liquid in tube as some backflow will occur and if it fills completely then fluid will be forced into the sample pressurization holes into the air line and clog the hydrophobic filter Troubleshooting Fluid levels The fluidics tanks are monitored by 6 sensors 1 One in the cube of sheath fluid detects when the 20 L cube is low and then empty 2 One in the sheath tank in the fluidics drawer determines when more sheath needs to be pum
17. o seeing lymphocytes in the center of the plot As such for better or worse your data will come under immediate scrutiny It s up to you but I would try to avoid this if possible The lymphs usually go in the left hand corner because it allows all cell population in PBL to be on the dot plot so you should do this as well Second adjust the instrument settings located under parameters and by changing increasing decreasing voltage This is your negative control tube so all cells in a dot plot should be in the left hand corner of the plot all histograms should be in the left side of the plot Adjust all parameters you have an actual positive stain in for instance if you are testing cell transfection with GFP and you put on GFP negative cells then worry ONLY about the FL1 setting if it looks like crap in the other channels don t worry you have nothing in those channels so it does not matter 2 Cells stained with the FITC antibody ONLY If your FITC antibody is of a very low percentage positive however let s say you use IFN g FITC for intracellular cytokine detection then use a different antibody that will give a healthy percentage of cells in this color say CD3 4 8 whatever You use this tube for FITC compensation DO NOT TOUCH VOLTAGES ONLY USE THE COMPENTATION BOX FROM HERE FORWARD Remember FITC can only overlap with PE so the only thing to adjust is the FL2 FL1 control which takes FL1 F
18. ometers once connected to a computer will not look for one again Thus it is necessary to start up the cytometer first wait 30 seconds and then start the computer If the computer is on and cytometer is off you must turn on the cytometer and then either restart or shutdown and restart the computer If the cytometer is on and the computer is off either being shut down or restarting from a crash you will need to turn off the cytometer wait 30 seconds then turn on the cytometer and then the computer If you do not see any events If it happened suddenly you were running fine and it stops make sure there is sample left in the tube and try taking the tube off wait a few seconds and then loading it again A clump of debris may have blocked the sample port and removing the tube allows sheath fluid to backflow and wash away the blockage If that doesn t work remove the tube again and press Prime which is a more forceful way to clear the sample probe it will switch back to standby after the cycle finishes Press run and try your tube again If you cannot see any events right from the start make sure the threshold is not set too high apple 3 and it should be set around 50 on FSC and your FSC settings are high enough for your particles If you still can t get events then you may have to restart the computer there are times when the computer will respond to changes to the instrument showing Ready or Standby in the stat
19. ped from the cube 3 One in the sheath tank in the fluidics drawer that tells the software when that tank is low empty 4 One in the waste cube that detects when the waste is full 5 One in the waste tank in the fluidics drawer that detects when waste needs to be pumped to the cube 6 One in the waste tank in the fluidics drawer that tells the software when that tank is full If the software gives you a not ready message after the warmup period check the status window apple 4 At the bottom will be a Sheath Empty or a Waste full message sensors 3 or 6 above This should not come up unless there is a problem with the fluidics cart Make sure it is turned on green power button on the right hand side f it is then make sure the warning lights on the left are not lit up and someone just silenced the alarm without changing the cubes If no lights are on then there is a problem with sensors 2 or 5 and you will need to find me If the waste alarm on the fluidics cart is going off and the tank is empty then you can try this Turn off the power to the fluidics cart green toggle switch remove the probe from the waste cube and dry it off with a paper towel or kimwipes including inside the hole in the grey cylinder towards the cap Return to the waste cube and then turn the power back on You may have to press restart to clear the alarm If it alarms again find me Macs look for attached devices when they are first starting up The cyt
20. ries sequential files Select all the files on the screen make sure all the boxes have a little box in the corner to denote this Go under plots and select next or previous to advance or go back a single file Or select change data file to change to a new data file somewhere else in your saved data You will also see a box for file increment increase This is usually set to 1 If you set it to another it will jump ahead or back that number when you select next or previous file That is if you are looking at file 001 and set the increment to 3 then select next file it will not go to 002 but to 004 Sometime people want to present data as a presentation To do this make the individual plots histograms EXACTLY how you want them to look Select copy and paste for export paste into power point files Printing To print the data on screen go to file select print You can also select print one which is a nice feature that will print one copy of all open pages you have on the screen If you have 3 pages open though and data on only one page do not use this feature it will print ALL pages and just waste 2 blank pages FACS Calibur Status Readings No tube Volt 10 tube on high 6 1v 5 5 6 1 med 6 3 lo 6 4 V 5 9 6 5 no sample tube move arm to side SV exactly 10 23V Power 15 no tube 2 5 if not 10 23 check for air leaks If FL3 and FL4 will not compensate try t
21. uctory class dates are announced on the training page go through the user manuals they are on the Calibur Mac in the Cellquest Pro folder or linked from the training page on our website observe another person operate the instrument at least twice schedule time with me to run at least your first experiment with you Location The FACSCalibur is located in CNY 149 room 5228 Take the central elevators to the 5 floor go through the door to the left of the balcony and then go through the door straight ahead to enter the lab Walk straight ahead and you will see the instrument in the bay in front of you When booking time for the calibur If you decide to not use your time or will not be using the entire time reserved please update or delete your entry as soon as possible so that other researchers will know it is available for use If you fail to delete your unwanted reservation at least 1 hour before the start time and do not use your time you may still be billed for 1 hour usage If you do not show up within 20 minutes of your start time your reservation is forfeited and anyone can use the instrument Researchers that are not members of the Partners AIDS Research Center Ragon Institute may not schedule reservations more than 1 week in advance There is a 2 hour limit during working hours 9 6 and no more than 2 reservations per week can be between 2 and 6 pm Delete unwanted reservation 1 hour before start time or you
22. urn your cd s You may also choose Open with Finder if the computer prompts you when you insert the blank cd and you simply drag and drop folders you wish to burn onto the cd icon and when finished drag cd to trash trash can will change to burn icon You can only burn to a cd once with this method and cannot add more files later How to burn Data CD On Cyto 2 1 Insert blank CD into the drive The CD eject button is in the top right corner of the keyboard the underlined triangle e click on the icon of the toaster for Adaptec Toast software in the dock e Create a new folder on the desktop and rename it whatever you want the cd to be named Move the folders files you wish to burn into this new folder The software makes the name of the cd whatever the first folder moved into it is called and all files are put into that Files to burn must be on the computer being used to burn the cd e You can use lt apple gt I to see the size of the folder as you add files e When you have collected the files you wish to burn drag and drop the folder to the Toast window You can add or remove files at this point by clicking the Data button e Click Write CD e Choose between Write Session or Write disk e Ifyou are using a CD R you can only write to it once so I don t think it matters which you choose e If you are using a CD RW you will not be able to write to this disk again if you choose
23. us window but will not be able to acquire events until you restart Cyto 2 and Mac analysis station run Cellquest Pro Layouts generated using Cellquest can be opened in Pro and data files can be opened in almost any flow software but layouts histograms dot plots and stats created in Pro CANNOT be opened in Cellquest for Mac OS 9 user manual on shelf contains a chapter on troubleshooting if I am not around __training manual good to look over chapter on cellquest etc 3 1 07 Protocol to prepare flow cytometer for acquisition of samples Note This protocol can not be used for DNA acquisition and or apoptosis assays 1 Ifcytometer and computer are both off Turn on cytometer by depressing large green button on side of cytometer right Let cytometer warm up for at least one minute before turning on computer 2 If cytometer is off but computer is on Turn on cytometer as directed above in 1 After 2 3 minutes restart the computer from Apple menu in the top left corner 3 If cytometer and computer are already connected Proceed with below The program used to both acquire and analyze data on the cytometer is called CellQuest Pro and the computer runs OS X This program must be running for all applications on this machine To launch CellQuest Pro click on the grey circle icon on the dock at the bottom of the screen Connect to cytometer go under the acquisition menu and select connect to cytometer Your
24. you gate the lymphocytes and look at say CD3 the percentage of CD3 cells in R1 will be greater than ungated total because you ve selectively enriched for this population with your gate Quadrant or histogram statistics Now put a quadrant on the dot plot to do this look at the palette and click the icon that looks like a window Now click the dot plot and drag the mouse The axes will appear These can be moved etc by clicking the crosshairs and dragging to the desired location To get quadrant statistics to appear with the desired plot selected border highlighted go to under stats and choose quadrant stats Also to put a region on a histogram select the histogram go to the palette choose the line with end lines looks like a long H put cursor onto histogram and drag it Editing statistics box information You can edit what information appears in the statistics box as well let s say for instance you only care about one percentage quadrant or something or are trying to squish a bunch of plots on a single page and the stats box size is prohibiting this then edit the box and what appears in it To do this go back to under statistics and choose edit quadrant stats Click off what you do not want and close when done This should apply to all stats now that you make in this document until it is changed again or the document is closed don t worry when you close your files it will not
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