Flow Cytometry: Immunofluorescence Staining of Activated and Resting Human Platelets

Flow Cytometry: Immunofluorescence Staining of Activated and Resting Human Platelets

PROCEDURE for preparation of activated platelets

1. Centrifuge tube of freshly drawn HEP or EDTA blood @ 600 rpm (75xg) for 20 minutes.

2. Remove all of platelet rich plasma (top layer) and place in clear 15 ml conical tube.

3. Wash platelets two times in PBS washing solution and resuspend platelets in PBS.

4. Add thrombin to the cell suspension to achieve the final concentration of 0.1 to 0.2 U/ml. Incubate at room temperature for 10 minutes

5. Add equal volume of 2% formaldehyde to fix platelets for 30 minutes at room temperature.

6. Wash two times in PBS and resuspend cell pellet in PBS. Cells may be stored at 4°C for up to 8 hours.

7. Stain activated platelets by direct immunofluorescence using CD62P-FITC, catalog number 555523 (31794X), at 20 µl/test, and compare with resting platelets from same donor.

ACCEPTANCE CRITERIA: Following activation, the CD62P positive reactivity of activated platelets must be >70% and negative on resting platelets.

PROCEDURE for preparation of resting platelets

1. Centrifuge tube of freshly drawn HEP or EDTA blood at 600 rpm (75xg) for 20 minutes. Note: The blood must be fresh and non-racked.

2. Remove all of the platelet-rich plasma (top layer) and place in a clear labeled 15 ml conical tube.

3. Add equal volume of 2% formaldehyde and mix gently.

4. Indicate the source of platelets (i.e. donor).

5. Keep at room temperature for 10 minutes.

6. Add approximately 10 ml washing solution (PBS+1%FBS).

7. Centrifuge at 2000 rpm (750xg) for 10 minutes.

8. Aspirate the supernatant and resuspend the pellet with ~10 ml wash buffer and centrifuge at 2000 rpm (750xg) for 10 minutes.

9. Aspirate the supernatant and resuspend the pellet in 6-8 ml washing solution (PBS+1% FBS).

10. Store fixed platelets at 4°C for up to 8 hours.

11. Use 100µl/test tube of platelets.

PROCEDURE for Flow Cytometric Analysis-Human Platelets

1. Add 100 µl of platelet suspension to the bottom of each tube.

2. Add the appropriate antibody to each tube.

3. Shake gently and incubate in the dark at RT for 20-30 minutes.

4. Remove tubes from dark chamber and vortex. Add 2 mls of washing solution to each tube.

5. Centrifuge for 5 minutes at 2000 rpm.

6. Remove the supernatant by aspiration, vortex and add 2 mls washing solution to each tube.

7. Centrifuge for 5 minutes at 2000 rpm. NOTE: If staining with a fluorochrome-conjugated primary antibody, proceed to step 14, otherwise, proceed to step 8.

8. Remove the supernatant by aspiration and vortex.

9. Add appropriate second step reagent to each tube and vortex gently.

10. Incubate in the dark at RT for 20-30 minutes.

11. Remove from the dark.

12. Vortex and add 2 ml washing solution to each tube.

13. Centrifuge for 5 minutes at 2000 rpm.

14. Remove the supernatant by aspiration.

15. Add 500 µl wash buffer to each tube and vortex. Platelets can be stored in 2% paraformaldehye at 2-8 oC for up to 36 hours prior to analysis.

SOLUTIONS:
Washing Solution: PBS + 0.1% Sodium Azide + 1% FBS.
Formaldehyde Buffer: 2% solution in PBS