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Indirect Immunofluorescence
Indirect Immunofluorescence Staining of Human Platelets
Procedure for preparation of activated platelets
- Centrifuge tube of freshly drawn Sodium Citrate, HEP, or EDTA blood @ 1000 rpm for 10 minutes.
- 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.
- 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.
- Repeat step 3 once.
- 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.
- Add equal volume of 2% formaldehyde to fix platelets for 30 minutes at room temperature.
- 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.
- 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
- 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.
- Transfer all of platelet rich plasma (top layer) to a new tube and mix with 10 µM PGE 1 as final concentration.
- Add equal volume of 2% formaldehyde to fix platelets for 10 minutes at room temperature.
- Wash platelets two times in PBS washing solution and resuspend platelets in the same solution.
Procedure for Flow Cytometric Analysis-Human Platelets
- Add 100 µl of platelet suspension to the bottom of each tube.
- Add the appropriate antibody to each tube.
- Shake gently and incubate in the dark at RT for 20-30 minutes.
- Remove tubes from dark chamber and vortex. Add 2 mls of PBS washing solution to each tube.
- Centrifuge for 5 minutes at 2000 rpm.
- 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.
- Add appropriate second step reagent to each tube and vortex gently.
- Incubate in the dark at RT for 20-30 minutes.
- Remove from the dark.
- Vortex and add 2 ml washing solution to each tube.
- Centrifuge for 5 minutes at 2000 rpm.
- Remove the supernatant by aspiration.
- 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
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