Flow cytometry program

Flow cytometry program

one. Boot procedure

1. Check the regulator power supply, turn on the power, and stabilize for 5 minutes.

2. Open the reservoir, drain the waste, and add 400 ml of bleach to the waste container. Open the pressure valve, remove the sheath tank, and add the sheath tank to 4/5 full (usually three steamed water, do PBS or FACSFlow for sorting), and close the pressure valve. Do sure to close the lid and check that all tubing is properly placed.

3. Turn on the FACSCalibur switch. The display of the instrument function control should be STANDBY and warm up for 5-10 minutes. Drain the air bubbles inside the filter.

4. If you need to print, turn on the printer.

5. Turn on the computer and wait for the standard Apple logo to appear on the screen.

6. Perform the instrument PRIME function once to exclude air bubbles from the Flow cell.

7. When analyzing the sample, first perform HIGH RUN with FACAFlow or PBS for about 2 minutes.

Note: After sorting, flush the pipe after each boot: install two 50ml centrifuge tubes on the sorting device, do not connect the concentration system, and press the white button in the lower right corner to start flushing. After the automatic stop, turn on the concentrating device and rinse it once with the above method.

two. Default Acquisition File (Acquisition Template Files)

1. Select CELLQuest from the Apple logo to see a new window that you can use to edit a get mode file.

2. Select one or more Dot Plots by selecting Dot plot from the drawing tool in the left column of the screen. Select Acquisition as the source of the graphical data from the Dot Plot dialog and determine the appropriate x-axis and y-axis parameters.

3. Select Histogram in the drawing tool in the left column of the screen, and draw the Histogram in the same way as above.

4. Name this window and store it in the FACStation G3\BD Applications \CELLQuestFolder \EXP folder, which can be called directly the next time you perform the same experiment.

Note: Two mode files have been set in this computer: ACQ and EXP, stored in the FACStation G3\BDApplications\CELLQuest \EXP folder, ACQ for cell DNA detection, and EXP for cell surface marker analysis.

three. Instrument setup and adjustment with CELLQuest

1. Select CELLQuest from the Apple screen, enter CELLQuest and open the appropriate acquisition mode file in the File command bar.

2. From the Acquire command bar at the top of the screen, select Connect to Cytometer (shortcut: +B) to connect the computer to the instrument. Move the Acquisiton Control dialog that appears to the appropriate location.

3. From the Cytometer command bar, open the four dialogs Detectors/Amps, Threshold, Compensation, Status, etc., and move them to the right of the screen to adjust the acquisition conditions at any time when you get the data. You can also get these four dialogs with +1, 2, 3, and 4.

4. In the Detectors/Amps dialog, first select the appropriate override mode for each parameter: linear mode Lin or log mode Log. In general, cell surface antigen analysis, such as analysis of peripheral blood lymphocyte subsets, FSC and SSC are mostly measured in linear mode Lin, and DDM Param selects FL2, while FL1, FL2 and FL3 are measured in logarithmic mode Log; analysis of cellular DNA When the content is FSC, SSC, FL1, FL2, FL3 are measured by Lin, and DDM Param selects FL2; when analyzing platelet phenotype, FSC, SSC, FL1, FL2, FL3, etc. are all measured by Log.

5. Place the sample to be tested, set the flow cytometer to RUN, and the flow rate can be HIGH or LOW.

6. In the Acquisiton Control dialog, select Acquire to start getting the cells. Select Pause and Restart at any time during the instrument adjustment process to observe the adjustment effect. Do not remove the " " before SETUP until it is fully adjusted.

7. In the Detectors/Amps dialog, adjust the signal multiplications in the FSC and SSC detectors: PMT voltages (rough adjustment) and Amp Gains (fine adjustment) so that the sample signal appears in the FSC-SSC dot plot and the three groups of cells Reasonable distribution.

8. Select the appropriate parameter setting Threshold in the Threshold dialog and adjust the Threshold level to reduce noise signals (cell debris). Generally, FSC-H is used for cell phenotype and FL2-H is used for DNA. Threshold does not affect the detector's acquisition of the signal, but it improves picture quality.

9. Select Region from the drawing tool in the left column of the screen and set the area line around the target cells, the so-called door. Determining the right cell population makes instrument adjustment easier.

10. In the Detectors/Amps dialog, adjust the multiplication of the fluorescence detectors (FL1, FL2, FL3, FL4, etc.). The cell population was adjusted according to the fluorescent negative control sample used to distribute it in the correct region.

11. In the Compensation dialog, adjust the fluorescence compensation required for two-color (or multi-color) fluorescent staining according to the standard fluorescence sample used. For example, if the cell population that should be FL1+ FL2- is distributed in the FL1+ FL2+ region, then FL2- needs to be increased? "?" in %FL1 and observe if the new adjustment is appropriate from the FL1-FL2 dot plot

12. In the Status dialog box: Laser Power: normal value—Run/Ready is 14.7mW, Standby is 5mW; Laser current: Normal value is around 6Amps.

13. The adjusted instrument settings can be stored in the Instrument Settings dialog box, and can be used for the next experiment.

Note: There are three parameter files in this computer that are stored in FACStation G3 \BD Applications\CELLQuest Folder \IMM-InstrSettings and FACStation G3 \BD Files\Instrument Settings Files \CalibFile and Mast-Cell-Set. For analysis of human leukocytes, the latter is used to analyze mouse mast cells.

four. Sample analysis with preset acquisition mode files

1. Select CELLQUEST from the Apple logo. After the new window appears, select Open from the File command bar to open the default acquisition mode file.

2. From the Acquire command bar at the top of the screen, select Connect to Cytometer to connect your computer to the instrument. Move the Acquisiton Control dialog that appears to the appropriate location.

3. Select Instrument Settings from the Cytometer command bar, select Open in its dialog box to bring up the instrument settings for the same experiment that was previously stored, and press Set to confirm.

4. In the Acquire command bar, select Acquisition&Storage to determine the number of cells, parameters, and number of channels to store. Among them, Resolution selects 256 when making cell surface markers, and 1024 when making DNA. Parameter Saved... selects different parameters according to different detection objects.

5. In the Acquire command bar, select Parameter Description to determine the file storage location (folder), file name (file), sample code and the parameters of the various parameters, that is, the detection parameters of the tube1, 2, 3... Generally, the data acquired by this instrument is stored in the FACStation G3\BD Applications \CELLQuest\IMM and DNA folders according to the detection object. The file is named according to the date.

6. In the Cytometer command bar, select Counters and move this dialog to the appropriate location to view the events count at any time.

7. Place the sample tube in the test area and select Acquire in the Acquire Control dialog to initiate the sample analysis.

8. Fine-tune the instrument settings. After the cell population is properly distributed, select Pause, Abort in the Acquire Control dialog box to remove the “ ” before Setup and start to officially acquire the signal and store the data.

9. When a certain number of cells are measured, the acquisition stops automatically and the data is automatically stored. Repeat step 7 to continue analyzing the next sample until all sample data has been analyzed.

Note: When all samples have been analyzed and analyzed, replace the three distilled water and place the flow cytometer in the “STANDBY” state to protect the laser tube.

Fives. Shutdown procedure:

1. Select Quit from File, exit the software, and select Don't Save to the Apple screen.

2. 4 ml of 1:10 diluted bleach was used as a sample, the sample was placed in a side by (vacuum is on), the outer tube was aspirated to about 2 ml, the sample holder was placed in the middle (vacuum is off), and then HIGHRUN for 5 minutes ( The inner tube sucks 2 ml).

3. Change to 4 ml of distilled water to make a sample and treat it as above.

4. Press Prime three times.

5. At this point, the instrument automatically switches to the STANDBY state and changes to 2ml of three distilled water. The computer, printer, main unit, and regulated power supply must be turned off 10 minutes after the instrument is in the “STANDBY” state to extend the life of the laser tube and ensure the normal operation of the application software.

6. Fill out the registration form.