KOSEN 한인과학기술자네트워크

Flow Cytometry Protocol for Human Peripheral Blood Mononuclear Cells (PBMC)

A. Solutions and Reagents

1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.

2. Formaldehyde (methanol free).

3. 2% Formaldehyde (stock formaldehyde diluted in PBS; make fresh on day of experiment).

4. Triton™ X-100.

5. Ficoll-Paque™.

6. Incubation Buffer: Dissolve 0.5 g bovine serum albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.

B. Fixation

1. Isolate PBMCs from whole blood by Ficoll density centrifugation as per manufacturer’s instructions.

2. Wash 2x by centrifugation using Incubation Buffer.

3. Resuspend cells in Incubation Buffer and aliquot at a concentration of 1 x 106 cells/100 µl/sample tube.

4. Aspirate supernatant and resuspend cells in 500 µl of 2% formaldehyde.

5. Fix for 30 min at room temperature.

6. Wash 2X by centrifugation in Incubation Buffer.

C. Permeabilization

1. Resuspend cells in 1 ml of 0.1% Triton™ X-100 (v/v in PBS).

2. Let stand for 30 min at room temperature.

3. Wash 2X by centrifugation in Incubation Buffer.

D. Immunostaining

1. Resuspend cell pellets in 100 µl of Antibody working solution (stock antibody diluted 1:200 in Incubation Buffer).

2. Incubate for 1 hr at room temperature.

3. Wash 2X by centrifugation in Incubation Buffer.

4. If using a fluorochrome-conjugated primary antibody, resuspend cells in 500 µl of Incubation Buffer and analyze on flow cytometer; for unconjugated or biotinylated primary antibodies, proceed to Step 5.

5. Resuspend cells in fluorochrome-conjugated secondary antibody or fluorochrome-conjugated avidin, diluted in Incubation Buffer at the recommended dilution.

6. Incubate for 30 min at room temperature.

7. Wash 2X by centrifugation in Incubation Buffer.

8. Resuspend cells in 500 µl of Incubation Buffer and analyze on flow cytometer.