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Flow Cytometry: Immunofluorescence Staining of Activated and Resting Human Platelets

PROCEDURE for preparation of activated platelets

1. Centrifuge tube of freshly drawn Sodium Citrate, HEP, or EDTA blood @ 1000 rpm for 10 minutes.

2. Remove all of platelet rich plasma (top layer) and place on the top of 0.5 ml of 30% human albumin in a plastic tube, centrifuge at 2000 rpm for 15 min.

3. Aspirate the supernatant and suspend the pellet with 10 ml of platelet washing solution, add 0.5 ml of 30% human albumin to the bottom, centrifuge at 2000 rpm for 15 min.

4. Repeat step 3 once.

5. Add thrombin to the cell suspension to achieve the final concentration of 0.1 to 1.0 U/ml. Incubate at room temperature for 10 minutes to activate platelets.

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

7. Wash platelets two times in PBS washing solution and resuspend platelets in the same solution. Cells may be stored at 4°C for up to 8 hours.

8. Stain activated platelets by direct immunofluorescence using CD62P-FITC, cat. no. 555523, at 20 µl/test, and compare with resting platelets from same donor.

PROCEDURE for preparation of resting platelets

1. Centrifuge tube of freshly drawn Sodium Citrate, HEP, or EDTA blood @ 1000 rpm for 10 minutes. Note: The blood must be fresh and non-racked.

2. Transfer all of platelet rich plasma (top layer) to a new tube and mix with 10 µM PGE₁ as final concentration.

3. Add equal volume of 2% formaldehyde to fix platelets for 10 minutes at room temperature.

4. Wash platelets two times in PBS washing solution and resuspend platelets in the same solution.

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 PBS washing solution to each tube.

5. Centrifuge for 5 minutes at 2000 rpm.

6. Remove the supernatant by aspiration and vortex. NOTE: If staining with a fluorochrome-conjugated primary antibody, proceed to step 13, otherwise, proceed to step 7.

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

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

9. Remove from the dark.

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

11. Centrifuge for 5 minutes at 2000 rpm.

12. Remove the supernatant by aspiration.

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

ACCEPTANCE CRITERIA: Following activation, the CD62P should be positive on all activated platelets and negative on the resting platelets.

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