流式细胞仪使用说明

FACS for fluorescently labeled cells

Prepare some cold sterile PBS in fridge before starting.

1. Cells were treated with the polymersomes/drugs for certain time before the experiment. When ready, cells should reach ~80% confluent to be sure that they are growing in log phase, a better stage for cell viability.

2. Remove the culture medium and wash cells with sterile 1x PBS once.

3. Detach cells with T/E or cell scrapper, the more gentle way to keep cells alive.

4. Collect cells in well-labeled 1.5mL eppendorf tubes and wash 1~2 times in PBS at 2,500rpm x 3min.

5. Remove the supernatant by using a 1,000mL tip and an additional 10uL white tip on it. Resuspend the cell pellets in certain amount of cold PBS, and pipette 100uL~150uL of the cell suspension into each well in a 96-well plate.

Note:

1. If you need to stain cells with primary and secondary antibodies to gain the fluorescence, follow the protocol for immuno-staining after step 4. After removing the 2nd Ab by centrifugation, continue to step 5. Fixation is NOT suggested.

2. Normally, we run the program as 10,000 events (10,000 cell individuals in theory) in 50uLs of “FACS-flow” (cold PBS in this case). So when resuspending cell pellets in cold PBS,

estimate the cell number you may have in each tube first. Too high cell number actually affects the FACS analysis or it may block the detecting probe.

3. Some companies provide their own sheath fluid instead of cold PBS to obtain single cell suspension.

4. If using a flow cytometry, cell samples are normally prepared in a glass tube (no lid). In this case, there is no need to remove all supernatant. But you need to know how much left there. So on such tubes, you may be able to find a label for certain amount of solution.