USER GUIDE TO BECKMAN COULTER FC500

Transcription

1 USER GUIDE TO BECKMAN COULTER FC500 Create a New Protocol Purpose: This procedure describes how to define the parameters and create rough analysis plots for a new experimental set-up. Some protocol templates with 1, 2, 3, 4, or 5 fluorescent parameters exist in the CXP/Users/FLOW/AcquisitionProtocols folder, which can be modified instead of creating a new protocol from scratch. 1. Open CXP Software and log in 2. Click File, then New, then New Protocol 3. Click Cytometer then Cytometer Controls 4. In the Acq. Setup tab, adjust the Discriminator, as needed (most mammalian cells = FS ; bacteria = FS 2-5). Also adjust Max Events, Duration, Red Laser Shutter, Live Gate, and Dots as needed. 5. Click Parameters. Select the desired parameters by checking or unchecking log or lin. Order of parameters can be altered in the Selected Signals Box. 6. Click OK. The program will prompt you to rename the protocol. Type in the desired name, then click OK. Leave the Cytometer Controls window open for future use. 7. Name the Axes: Click File then Edit FCS Header Attributes. Click on FL1 and type in the fluorochrome for it Name. Continue with FL2, etc. Click OK when finished with naming. 8. Create Plots: Dot plots and/or histograms with appropriate axes by selecting the desired plot icon from the toolbar or by clicking Plots and then choosing the desired plot. Select the appropriate parameter for each axis. 9. To easily arrange the plots, click Window, then Tile Special, then Large (or adjust tiling strategy as desired). 10. Basic regions can now be created using icons from the toolbar or by clicking Analysis, then Create Region and then choosing the desired region. You must first click on the plot upon which you would like to create the region before you may click on an icon from the toolbar. 11. To apply a region as a gate on a plot, right-click on the plot, then select Format Plot. In the Data Source tab, select the appropriate region from the Gate dropdown. If you wish to apply the region as a gate to multiple plots, the fastest way is to check Apply gate to all plots. To remove the gate from plots that do not require the gate, right-click, select Format Plot, and change the Gate drop-down to Ungated. 12. To create an automatic stop when a certain number of cells are displayed on a specific plot, right-click on the plot, then select Format Plot. In the Stop and Save tab, check Use Stop Condition, then enter the desired number. 13. Additional or fewer statistics can be reported. Select Analysis, then Select Results. Select the statistics that are desired. 14. Appropriate voltages and compensation should be determined for each parameter using a sample of unstained (or otherwise non-fluorescent) particles, as well as the required controls (see section on Running Samples, Single Tube mode). Regions should also be adjusted. Save the protocol after any change is made!!!

2 15. Select the appropriate Flow Rate from the drop-down menu (recommended: Low or Medium). Changing Axis Name Purpose: It may be desirable to change the word(s) on an axis. The steps below describe how to input the axis name so that it shows on the graphs and gets stored in the LMD file. Things to know: -The Parameters section of the Cytometer Controls window is where you can give a name to the Detector Name term. This may guide you when adjusting voltage settings and viewing the Parameters in the Cytometer Controls window, but the name does not get stored anywhere that will show up on plots in CXP, FlowJo, etc. -You cannot change the $Parameter term, which makes up the first part of the axis name in the LMD file. -Under FCS Header Attributes is where you can change the name of the $Stain term. This makes up the second part of the axis name when the file is opened in FlowJo (FCS2.0 format). If you type FITC in the $Stain box for FL1, the name will look something like this in FlowJo: FL1:: FITC -By default, plots in CXP software use the $Stain term as the axis labels. If you would rather have it show the $Parameter or another term, this can be adjusted in Labeling tab of the Format Plot window (right-click on a plot and select Format Plot) *If you are unsure which detector is being displayed on a given plot, click on the plot and hover the mouse over the axis name until >>> is displayed. Click. *In the window that opens, click on the Labeling tab. *Find the appropriate axis (X-axis or Y-axis) and select $Parameter from the dropdown list. *Click OK. Now the axis should be labeled with the detector number (FL1, FL2, etc.) *When you know which detector you would like to associate with a certain name (perhaps the fluorescent molecule name, the antigen which the fluorescence in that detector represents, etc.), click File. *Click Edit FCS Header Attributes. *In the window that opens, in the Parameters section locate the detector which you would like to associate with a name. Click on that detector s name (i.e. FL1 log, FL1 lin, FL2 log, FL3 log, etc.). *On the right side of the window locate Name and click in the textbox below it. *Type in the name you wish to show on the axis. *Click OK. *To make $Stain display as the axis name in a plot, open the Format Plot window a. Right-click inside a plot and click Format Plot

3 OR b. Click on the plot and hover the mouse over the axis name until >>> is displayed. Click. *In the window that opens, click on the Labeling tab. *Find the appropriate axis (X-axis or Y-axis) and select $Stain from the dropdown list. *Click OK. Now the axis should be labeled with the name you assigned to it. *Save the protocol after making any of the above changes to make the changes permanent.

4 Naming LMD Files Purpose: At each use of the software, adjustment of the User s settings controls how LMD files will be named. This procedure describes multiple ways to set up the Workspace Preferences to achieve the desired naming strategy. A. General Guidelines 1. LMD filenames are controlled in the Workspace Preferences. To get to this, click File, then Workspace Preferences, or press CTRL+W. 2. Workspace Preferences are NOT linked to a protocol; they are linked to a User. This means if a person sets the Preferences but the Preferences are changed by another individual logged in as the same User, the Preferences originally set by the first person will not be present when he/she opens his/her desired protocol. 3. There are up to 4 user-input parts to a LMD file name: Sample ID1, Sample ID2, etc. Many other automatic parts are also available, including the date, time, run number, and protocol name. 4. When running in carousel mode, LMD file name MUST include both Sample ID1 & Sample ID2, in addition to other parts desired. When running a sample in Single-Tube mode, LMD file name only needs to have Run Number.**** B. To generate filenames like: YourName SampleName Run#.LMD 1. Set Workspace Preferences (see A1 above) as follows i. If running in Automatic mode 1. In LMD File Name tab, check Sample ID1, Sample ID2, Run # 2. In Acquisition Options tab, uncheck Edit Sample ID (this will prevent the software from asking for manual entry of the sample name after each tube is completed) ii. If running in Single Tube mode 1. In LMD File Name tab, check Sample ID1, Sample ID2, Run # 2. In Acquisition Options tab, check Edit Sample ID (this will allow the user to manually enter the sample name after each tube is completed) 3. When the sample stops running, the computer will prompt for editing. In the first box, Sample ID1, enter name (YourName above). In the second box, Sample ID2, enter sample name (SampleName above). C. To generate filenames like: SingleComponent.LMD 1. May not do so in Automatic mode. Automatic mode requires that SampleID1 and Sample ID2 be utilized.

5 2. If running in Single Tube mode, set Workspace Preferences (see A1 above) as follows i. In LMD File Name tab, uncheck all boxes except for Sample ID1 1. If a different component that is not manually entered for each sample (i.e. Run #) is desired, check that desired component instead of Sample ID1 ii. In Acquisition Options tab, check Edit Sample ID if manual entry of the sample name is desired iii. When the sample stops running, the computer will prompt for editing. In the first box, Sample ID1, enter sample name

6 Running Tubes A. Single Tube Mode allows the user to place one tube at a time onto the carousel. The sample is run, then the instrument stops & returns the tube. The first tube must be removed and then another tube can be loaded in the same position. All tubes are placed in position 10 of the carousel, which can be accessed by the small Tube Access door on the front of the carousel lid when the instrument is in Single Tube mode. When in Single Tube mode, the lid of the carousel should not be opened. 1. Adjust the Workspace Preferences, if needed, to produce the desired naming and printing results. 2. Click on the Single Tube button in the toolbar. 3. Lift the Tube Access door, insert tube, and close door. 4. Click on the Run button in the toolbar. i. While running, voltages and compensation values can be altered from the Detectors and Compensation tabs, respectively, on the Cytometer Controls window, accessed by the button on the toolbar or by clicking Cytometer then Cytometer Controls. ii. If setting up a new protocol, it is advisable to alter the voltages/compensation under Setup mode, which can be activated through a checkbox in the Parameters tab of Cytometer Controls. 5. After starting the run, the software will display a series of messages at the bottom of the screen: Awaiting Sample, Preparing Sample, Acquiring, and Stopping. When complete, the tube will return to the Tube Access door and the software will display the Awaiting Sample message.

7 B. Automatic Mode allows the user to load up to 32 sample tubes into the carousel and program the instrument to automatically run each tube in sequence. Automatic Mode is the default setting for the CXP Software; no special button must be pressed to enable Automatic Mode. 1. Adjust the Workspace Preferences, if needed, to produce the desired naming and printing results (see Naming LMD Files and Setting Printing Options ). 2. The Acquisition Manager window must be visible. If it is not, click View and then Acquisition Manager. 3. If samples were being run in Single Tube mode ( clicked and shaded), click again to take it out of Single Tube mode. 4. Many people find it helpful to change the display of the Acquisition Manager window. To do so, click View, then Customize Worklist Columns. Uncheck Tube ID. Check Sample ID2. Adjust column size in the Acquisition Manager window. 5. Click the Clear Worklist icon in the toolbar. 6. For each sample being run, click on the Insert Test icon in the toolbar. 7. In the Acquisition Manager window, enter what is desired in the Sample ID1 column. Hit Enter, and this text will be added to each test and cannot be changed for each individual test. 8. Here is where organization is essential! In Sample ID2, enter the specimen names. When entering the first sample name, after hitting Enter the software applies this name to all of the tests. Simply use the down arrow key and type the second sample name, then down arrow and type the third, etc. The software should allow you to simply overwrite. 9. Lift the lid of the carousel and load sample tubes into the appropriate numbered slots. Ensure that the sample name entered into the Acquisition Manager worklist corresponds to the sample tube in the corresponding numbered slot in the carousel. 10. Examine the carousel and identify which number it is (1 or 2) by the white writing on the top knob. Type the number into the Carousel Number column of test 1 of the Acquisition Manager window, then hit Enter. 11. To run the carousel, click the Run button in the toolbar.

8 Quality Control check on the FC500 Every morning (Monday - Friday), Facility staff run 2 QC bead sets to ensure that the lasers & PMTs are functioning normally. The QC beads also check to make sure the lasers have "warmed up" enough and are functioning at the appropriate power level. Very rarely do we see results that are abnormal - and usually when we do the problem is either a) a bubble in the fluidics (solved by priming several times) -orb) some miscommunication between the computer & the cytometer (solved by shutting off the cytometer and restarting; or by shutting down the cytometer & computer and restarting. When this happens, after shutting down I use "FC On (Warmup)" to turn on the FC500, not the "CXP Cytometer" icon. Then, once the FC500 is on and the "laserready" arrow shows up on the front panel, I then open the CXP software). If you wish to run the QC beads to ensure accuracy of your results, please follow these instructions: 1. After turning on the FC500, wait for the "laser-ready" arrow to show up on the front panel. 2. After logging in to CXP software, open the "1A_beads " Acquisition Protocol under the FLOW user. 3. In the refrigerator (top shelf, left-hand side) is a flow sample tube with tape around it that says "FC500 & XL FlowCheck ". Vortex, then run this sample under the beads protocol. 4. A bunch of events should show up in the rectangular gate in the FS vs SS plot. Also, a peak should show up in each of the linear gates in the histrograms. If not, abort and then try priming twice. Try to run the tube again. If still no luck after more priming, shut down & restart. 5. Assuming the events & peaks show up, after starting the sample, RESTART after about 5 seconds. Then let the sample run til it automatically stops at 5000 events. 6. The HP X-CVs for each linear gate should be no more than The normal range for each FL channel is listed on the inside cover of the FC500 Log Book vol. 2 binder, sitting on top of the bookshelf on the other side of hte wall from the telephone. 7. If the HP X-CVs are outside of the acceptable range, prime and then run the tube again. Usually this fixes it. If the CVs remain high, something is wrong with the optics, laser(s) or fluidics, and you should contact a Facility staff member (phone numbers & s will be posted by the cytometer).

9 8. After running the FlowCheck beads, find another tube in the refrigerator labeled "675 beads " Open the "1A_675 Beads " Acquisition Protocol under FLOW. Be sure that the numbers on the tube match up with the numbers in the Acquisition Protocol. 9. Run the sample as in 3-7. The HP X-CV values should be no more than Return the tubes to the same place you found them in the refrigerator. Put the printouts in the FC500 Log Book vol. 2 binder sitting on top of the bookshelf on the other side of the wall from the telephone. If the volume in either tube gets too low, locate the appropriate small bottle of beads in the refrigerator, right in front of where the tubes of beads are. The one with a blue dot on top & a pink line around it is the 675 Bead bottle; the one with the blue line is the FlowCheck bottle. VORTEX the bottle for 10 seconds, then put enough drops into the appropriate flow sample tube to bring up the volume. DO NOT TOUCH THE TIP TO ANY SURFACE! This will contaminate the beads & make them un-useable (a loss of several hundred dollars)

10 Setting Printing Options Purpose: Turn on/off printing of FlowPages. Some users wish to have these printouts; others do not. 1. Printing preferences are controlled in the Workspace Preferences. To get to this, click File, then Workspace Preferences, or press CTRL+W. 2. Workspace Preferences are NOT linked to a protocol; they are linked to a User. This means if a person sets the Preferences but the Preferences are changed by another individual logged in as the same User, the Preferences originally set by the first person will not be present when he/she opens his/her desired protocol. 3. Click on the Acquisition Options tab in the Workspace Preferences window. 4. To print the FlowPage, check the box next to Print FlowPages. To prevent printing, uncheck the box. 5. If a sample has already been run but printing was not turned on, it is possible to manually print the FlowPage. a. Click File, then Print. b. In the bottom-left, ensure that nothing but FlowPages is selected for printing (i.e. NOT histograms, plots, etc.) c. Click OK. 6. If a FlowPage has not been created for a protocol and printouts of the plots are desired, follow step 3 above, and in step 4 click the box next to Print Plots.**** 7. A way to save paper yet have a record of the results from the plots is to NOT print, but instead save the FlowPage as a PDF. This option is reached by following step 3 above, and in step 4 checking the box next to Print to PDF.****

11 Start-up Procedure: 1. Verify that the Sheath Fluid container is full. Fill with Sheath Fluid to the blue line. 2. Verify that the waste container is empty. If it contains waste, carefully dump into sink and add bleach to the first line (bleach is under sink) 3. Ensure that the printer is filled with paper. Fill with paper from the shelf if not. 4. Turn on printer with the switch on the back of the printer. 5. If the computer is not on, turn it on. If needed, log in to the computer (see yellow note on screen). 6. Turn on power to the monitor screens. 7. Turn on the cytometer by double-clicking the FC ON icon on the computer Desktop 8. The lasers will require about 15 minutes to warm up before the cytometer is ready to be used. The lasers are ready when a green arrow-head appears on the front display of the FC Use one of the workstation computers to log-in to RIMS: Double click and open the RIMS Login on the desktop and log-in a. Log in to RIMS using your PSU user ID and password b. Click on New Login under Lab Time and log in with your desired Project c. Back on the home screen, you can now click on your reservation time under Equipment My Reservations. Or, click on Use Equipment under Equipment. Log in with your desired Project d. It is okay to now log off of the RIMS website (and stay logged IN to the equipment) by clicking the door icon in the upper right corner. Or, it is also okay to remain logged in to the website. 10. Double click and open the CXP Cytometer icon on the Desktop, select the User and enter the Password (Password: password), then click Next 11. Click on a Protocol, if desired; then click Finish 12. Ensure that the cytometer is not in Idle mode; if it is, click on the Idle mode icon 13. Click the Prime icon. It may be necessary to click this icon multiple times before the instrument is ready to run, as sometimes an air bubble will get trapped in the flow cell during the shutdown/startup procedures. If an air bubble is trapped, the cells in the sample tube will flow through the cytometer but will not be detected. Thus, pay attention as the first sample tube is run to ensure that the cells are being detected.

12 Routine Cleaning for All users after each use: (please note: feel free to log out of RIMS before washing and/or shutdown you should only be charged for time you spend with your research, not cleaning!) 1. After finishing your data acquisition, clear the worklist by clicking You should see the Acquisition Manager clear. (If the Acquisition Manager is not visible, use View and select Acquisition Manager.) 2. Be sure that the software is in carousel Automatic mode. The cytometer is in carousel Automatic mode when the message Set Automatic Mode appears in the lower right corner of the software window. a. If samples were being run in Single Tube mode ( clicked and shaded), click again to take it out of Single Tube mode. b. If samples were being run in MCL Manual mode ( clicked and shaded), click again to take it out of MCL Manual mode. 3. Insert a new panel by clicking 4. In the window that appears, click on the drop-down list next to Look in and click on Users. Then click on FLOW, then Panel, then cleansepanel. Click Open 1. This will put 4 lines into the Acquisition Manager window. In the Carousel No. column, type in the number of the carousel being used (1 or 2) and push Enter. Ensure that the Location in the next column fills in with 1, 2, 3 and 4. See example to the right > 2. Put about 2mL of 10% bleach solution into a sample tube 3. Put about 2mL of distilled water into each of three sample tubes 4. Place the bleach tube in position 1 of the carousel; place the water tubes in positions 2, 3 & 4 5. Put the carousel into the MCL and close the MCL cover 6. Click 7. Wait. The software may ask if the protocol that was previously being run should be saved; answer Yes or No as desired. 8. Wait for the cytometer to automatically run all 4 tubes. It will take about 10 minutes.

13 9. When the cleaning panel is finished, the messages at the bottom of the software window will read Awaiting Sample and Set Automatic Mode 10. Remove the bleach and water tubes from the carousel 11. If shutting down instrument, proceed to Shutdown Procedure instructions. Shutdown Procedure: (all after-hours users should shut down the instrument after cleaning unless the next user is in the room) 1. After cleaning, press the Idle mode icon 2. Wait for the instrument to transition into Idle mode. The message at the bottom of the software screen will read Press Idle Mode button to Initialize 3. Fill the reservoir of each of the two black Cleaning Adapters with distilled water (about 5mL each) 4. Open the MCL cover and load the Cleaning Adapters into the carousel. a. Put the Cleaning Adapters into positions 1 and 2 of the carousel b. Position the filled reservoirs toward the center of the carousel 5. Put the carousel into the MCL and close the MCL cover 6. Ensure that the message at the bottom of the CXP software screen reads Press Idle Mode button to Initialize. Then, press the Auto-Cleanse icon 7. Wait for the Cleanse cycle to complete. 8. The message Cleanse cycle in progress appears during the cleaning cycle. When the cleaning cycle is done, the message changes back to Press Idle Mode button to Initialize. 9. When completed, press the Idle Mode icon 10. Exit the software (either File Exit) or click the large X in the corner 11. Turn off the FC500 by double-clicking FC OFF on the Desktop Data Transfer Procedure: 1. After finishing running your samples, close CXP software 2. Double click on the Micro_cyto workgroup icon below the tree icon on the desktop then look for the Fc500 icon shaped like a computer and double click on it 3. Go back to the RIMS login computer you initially logged in with, and find the Users (FC500) folder on the lower left hand side of the desktop and double click 4. A login window will appear. a. The login will be: Fc500/general b. The password is: four**** 5. To upload to the PASS space, open the browser and go to webfiles.psu.edu (other storage sites can be used like Box or the PASS space). For webfiles, your login and password will be your PSU ID and password. When you log in, on the left hand side you ll open the folder called PASS Personal, then upload your files

14 by finding your folder and data in the Users (FC500) folder (Should be under Desktop FC500 Users PI s Folder FCS files your data folder) 6. You have now transferred your data, you can log out and access it from your own computer by going back to webfiles.psu.edu (or your storage website)