For immunology, especially oncology experiments, it is inevitable to measure the killing of tumor cells by lymphocytes or other cells. The most commonly used foreigner is the Cr51 radiation experiment, but this method cannot be done in many places in China, and the MTT method It is too old, and it is not accurate, so the alternative method is to use flow cytometry, which is convenient and accurate. This is also the method of referring to foreigners.
1 Count the target cells and add CFSE to make the final CFSE concentration reach 2uM at 37 degrees for 30 minutes.
2 After taking out, 1000rpm, 5min, you can vaguely see the cells stained yellow, washed three times with PBS
3 Mix with effector cells for 4-6 hours.
4 Digest and take out the cell mixture, wash twice, PI working solution 5ul, avoid light in 30 minutes
5 CFSE, PI double positive cells are the target cells killed, divided by the total number of target cells, which is the killing rate.
Precautions:
1. The concentration of CFSE and PI must be paid attention to, rather than lower than high, personal feeling, if the level is higher, it seems better to put it at 4 degrees overnight
2. The reference of the target cells must be set for total negative, CFSE single standard, PI single standard, CFSE + PI double standard and synchronization.
3. The duration of action should not be too long, do not exceed 8 hours.
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