LSR II Users Guide Stanford Shared FACS Facility 06/10/13

Transcription

1 LSR II Users Guide Stanford Shared FACS Facility 06/10/13 A configuration sheet is located on the Website in the Instrument Overview area Do I need to run beads when I collect data using Diva Digital? For each data collection, you need to first collect data on 197 calibration beads. What samples do I need to bring? All users should always bring single stained compensation samples If using 3-color staining: User should bring 4 compensation tubes! Each single stained tube with one fluochrome AND one unstained tube. No PI or viability dye in the single color controls Don t have enough cells for compensation? User can purchase compensation beads from BD or Invitrogen BD CompBeads Anti-Rat/Hamster!for mouse samples BD CompBeads Anti-Mouse for almost all human samples PPE must be worn at all times when in our BSL-2 environment Lab coat, gloves, closed toed shoes, are mandatory, respirator and goggles are available

2 Starting Up the System Allow 30 minutes warm up time for the lasers 1) Turn on the Cytometer. The power switch is located on the lower right corner on the Cytometer. The fluid control button lights green and the Standby button lights amber. 2) SSFF computers are left on at all times. LSR-II 20L Pump System Instructions (Cytek) Fluid mangers are in use on all our LSR II s. Use offers the following advantages: 1) No need to open and fill the metal tank. 2) Stabilizing of sheath flow rates to allow a 6 usec window extension. To use: 1) The pump system turns on automatically with the LSR. However, it does have its own switch as well(on the.lsr II and LSR II.UV It is the blue box labeled Cytek under the LSR with the black lever switch, for the LSR II.2 it is the FACSFlow Supply System). Verify that this is on. The LSR will not work properly otherwise. 2) Make sure there is enough sheath in the cube for your experiment. The cube is on a scale, so you can look at the weight of the cube on the scale, remembering that 1 L weighs 2.2#. 3) If the sheath cube is empty replace with a new one (heavy so lift carefully) from the either the hall or the rear of the room. Note that the cube closure just screws on there is no pressurization of the cube. Be careful not to kink the tubing 4) Empty waste in sink in hallway when full, using the protective face shield, gloves and lab coat. Remember to add bleach to line marked on carboy. 4) Alarm sensors are connected to the cube and the waste, indicating when they need attention if you forget. The LSR won t run samples properly if the pump is turned off. The pump should never be turned off manually!! 5) Please [email protected] with any problems or questions Diva Software Instructions 1)Log into Diva software using BDFacs Diva software icon on desktop Username is Sunet ID/ password starts with a capitol letter 2) Watch for Instrument connected status in bottom of the Cytometer window 3) If CST mismatch dialog box appears select use CST settings Setting Up Experiment 1) Go to Experiment menu New Experiment Select the SSFF tab and choose the

3 newest date available select experiment Click! OK Rename - right-click!rename Use descriptive name and date 2)Edit menu!user Preferences!Select desired defaults. You can deselect save analysis and load data after acquisition to save time(buffer). Calibration 1) Run standard 197 calibration beads (located in rack or freezer) load sample on the SIP and hit RUN and LO buttons on Cytometer panel 2) Go to Beads Specimen (syringe icon) CLICK on + sign 3 ) Click on 197 Beads tube (green arrow! is pointing at the tube) This makes acquisition controls active In Acquisition Dashboard CLICK!Acquire Data 4) Set Events to Display to for calibration 5) Set flow rate to low 200 events per second. After beads appear on plots, use fine tune (black knob) to adjust flow rate to aprox 200 beads/sec 6) Check that the global work sheet is selected. (acquistion must be stopped in order to change the worksheets) 7 ) Follow the onscreen instructions and check the instruments laser delays, area scaling and MFI values are calibrated(located on second worksheet Standards). 8)If the delays are off first check the Current values, they are located on the desktop, use the latest performance shortcut for the most recent CST run. 9) On the first global worksheet check delay window extension a) Confirm the Forward scatter threshold is set to 50% of the forward scatter signal. B ) Check the laser delays by closing the window extension to 0. Open window extension to 6 when done. c) If properly set the area signal for the Green, Red and Violet lasers should drop no more than 50%.(you can use the height value for a quick reference to 100 %signal.) d) Check that Area Scaling values are correct. Area should be greater than or equal to the height within 10%. adjustments in the area scaling section in the laser tab.

4 e) Switch to the standards global worksheet and adjust the PMT voltages (cytometer window -> parameters tab) so the measured values are at or around displayed target values in bold(10%). f) Record data for beads in tube labeled 197 beads after calibration is complete. Experiment Definition 1) Under Experiment menu select New Specimen!Click on + to see tube 001 2) Highlight tube one in new specimen area >click on arrow on left 3) In the Cytometer pane, deselect parameters you will not be using. Do not delete Parameters that may be of interest. If you add back a deleted parameter to the experiment it will come in with different voltages not specified pmt settings by facility. Check cytometer settings for the 197 bead tube to see correct pmt voltage to assign. This may cause problems for compensation calculations In Parameters tab, click on small button to left of parameter name Click delete button (use shift key and highlight for multiple deletions) Repeat for each parameter you're not using 4) Keep forward and side scatter height and width selected 5) Lower forward scatter PMT for typical cells in the parameter tab 6)Lower the threshold for cell population in the threshold tab 7) The data may be viewed compensated or not, but the uncompensated data is written to the database along with the matrix so that other compensations may be used later in FlowJo. Note: For each tube collected in Diva software, uncompensated data is recorded and the compensation matrix is applied as a transform and not affect your data. Compensation All samples must be filtered for sorters 1. Select Experiment>Compensation Setup>Create compensation tubes. This will generate a new specimen in your experiment called Compensation Controls. Within the specimen it makes a tube and a worksheet for each compensation control, including one for unstained cells. 2. Run unstained cells first. Set Events to display to Adjust the FSC and SSC voltages to place the population of interest on scale. 4. Adjust the FSC threshold value to exclude most of the debris without excluding the population of interest.

5 5. Move p1 gate on FSC/SSC plot to include your cells of interest, then right-click and Apply to All Compensation Controls if all your controls are expected to fall in the same FSC v SSC gate. 6. Run each sample in the unstained cell tube area so we can define that all events are on scale, and that each stain is most dominant in its own channel (by a minimum ½ log). Keep in mind that if you lower the voltage below the baseline voltages defined for the instrument you may lose the ability to detect low positive events on that channel. Try to avoid lowering voltages below these target values. It is acceptable to have the negative peak shift above the first log decade in some detectors. 7. Put on your positively stained sample to verify that the positive peaks remain on scale. Lower voltages on those parameters that are off scale (place below 10 x 5). Remember, if you significantly lower the PMT voltage below the baseline settings in order to bring the positive population on scale, then the dim populations might not be easily resolved from the negative populations for that parameter. 8. Run the compensation samples and collect data for each in the appropriate tube. Use Next to move between tubes. For each tube, make sure the integral gate encompasses your positively stained cells. 9. Select Experiment>Compensation setup>calculate compensation. This calculates a compensation matrix. If the calculation is successful, a dialog appears for you to enter a name for the compensation setup. To keep track of compensation setups, include the experiment name, your initials, or the date in the setup name. Error messages that warn you of >100% compensation values indicate one of two things, the voltage settings used are suboptimal and a channel receiving less total photons has been over amplified or that the color combination at the settings you have chosen is not optimal. You can ignore these messages, but it would be best to try increasing or decreasing voltages on the problem channels and rerunning all your comp controls at new settings. You will have to recalculate compensation. If there are still problems, it may indicate that you do not have a good color combination. Cell analysis and Data Acquisition 1. Create a series of plots on the global worksheet that you will need to set up your acquisition. It is best to have at least one axis for each color minimum in case there is an issue with signal levels across samples. 2. Pull up a population hierarchy and make gates as needed for your sort or analysis. 3. High light and Select the first tube in specimen 2 and rename the tube with the proper sample ID 4. Load your tube and record the data 5. Save your analysis template for future use. Click on the analysis template and rename it, then export analysis template 6. Run bleach (blue top 10% bleach) for 2 minutes on high followed by water(black top)for 2 minutes on high. 7. Leave the machine in standby on the low setting with a tube of water on the SIP

6 You must check in your data to protect it Data is deleted monthly if it is not checked in it is gone USB and personal back up devises are not allowed on our machines Discs may be burned when alternatives are needed Data Transfer to FACSData Server When finished with data collection: 1)Select experiment to be exported for CHECKIN 2)Select File!Export!Experiments 3)Data Export Pathway C:\Export 4)Check that the proper experiment is being transferred to the folder area. Click OK. 5)On the Desktop, activate by double clicking!facs Data CHECKIN 6)The appropriate experiment should be displayed and selected. Click OK. The named of the person logged in needs to be selected on the list and the SUNet Id name should be displayed at the bottom. Click! OK. Add address to send the FlowJo summery file to additional people 7)A.jo file for MAC and a.wsp file for PC will be ed. 8)If it asks you something about delayed sessions, click! cancel. Note: The person s name in the first box becomes the owner 9)FlowJo PC users should install the WebStart Version of FlowJo PC. Start FlowJo PC and from the FileMenu select Open and then open your.wsp file. You will be prompted for Username: flowjo Password: Shutting Down the System Perform the following procedures at the end of each day or when required. fluidics cleaning system shutdown Always clean the BD LSR II Cytometer before it is turned off at the end of the day. Proper cleaning will ensure that your Cytometer will function consistently. Cleaning prevents the sample injection tube from becoming clogged and removes dyes that could remain in the tubing, causing carryover. Fluidics Cleaning Follow this procedure immediately after running viscous samples or nucleic acid dyes such as Hoechst, DAPI, propidium iodide (PI), acridine orange (AO), or thiazole orange (TO). It will take approximately 5-10 minutes to complete the shutdown procedure. NOTICE Make sure the sample fine adjust knob is at the middle flow rate position or greater for the entire cleaning procedure. 1 Set the fluid control to RUN. 2. Install a tube containing 1 ml of a bleach solution on the SIP,run for 3-5 minutes. Use our blue tube 1:10 dilution of bleach in DI water as the bleach solution. 3. Repeat steps 2 with DI water. 4. Leave a tube containing no more than 1 ml of DI water on the SIP. A tube with 1 ml of DI should remain on the SIP to prevent salt deposits from forming in the

7 injection tube. The tube also catches back drips from the flow cell. ~ CAUTION Do not leave more than 1 ml of water on the SIP. When the BD LSR II flow cytometer is turned off or left in standby mode, a small amount of fluid will drip back into the sample tube. If there is too much fluid in the tube, it could overflow and affect Cytometer performance. 5. Set the cytometer to standby mode. System Shutdown 1. Turn off the flow cytometer.

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