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11-2019

流式细胞术(FC)

发布时间:2019年11月13日

Flow Cytometry General Protocol

1. Buffer and solution preparation

a) Mix 1 portion of Fixation/Permeabilization Concentrate with 3 portions of Fixation/Permeabilization Diluent.

b) Prepare a 1X working solution of Permeabilization Buffer by mixing 1 portion 10X concentrate with 9 portions distilled water.

2 Aliquot 5X105-1X106 cells into each assay tube. (Disperse cells by gentle pipetting and filter it through a cell strainer)

3. Add 1mL of 1% FBS into PBS and then centrifuge the cell suspension at 2000 g for 4 minutes at 4℃. Discard the supernatant.

4. Repeat Step 3 for 2 times.

5. Resuspend the cell pellet into 200 µl of Fixation/Permeabilization Buffer. And then incubate it for 30 min at room temperature. Protect it from light.

6. Add 0.4 mL of 1X Permeabilization Buffer and then centrifuge the cell suspension at 2000 g for 5 minutes at room temperature. Discard the supernatant.

7. Repeat above Step 6.

8. Block it with 2% normal goat serum in 1X Permeabilization Buffer and then incubate it for 15 min at room temperature.

9. Without washing, and directly add the recommended amount of antibody for detection of intracellular antigen(s) to cells and  then incubate it for 60 min at room temperature.

10.Add 0.3 mL of 1X Permeabilization Buffer and then centrifuge the cell suspension at 2000 g for 5 minutes at room temperature. Discard the supernatant.

11. Repeat above Step 10.

12. Add 50uL of the diluted secondary antibody to each tube. And then incubate it for 30 min at room temperature. Protect it from light.

13. Add 0.4 mL of 1X Permeabilization Buffer and then centrifuge the cell suspension at 2000 g for 5 minutes at room temperature. Discard the supernatant.

14. Repeat above Step 13.

15. Resuspend the cells in 0.3 mL PBS and then have it analyzed on flow cytometer.

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