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P r o t o c o l s

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 4C for up to 8 hours.

7. Stain activated platelets by direct immunofluorescence using CD62P-FITC, catalog number 555523 (31794X), at 20 l/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 4C for up to 8 hours.

11. Use 100l/test tube of platelets.

 

PROCEDURE for Flow Cytometric Analysis-Human Platelets

1. Add 100 l 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 l 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

 

 
Protocols Index>>
Flow Cytometry
Fluorochrome Absorption and Emission Spectra
Procedure for Setting Compensation for Multi-Color Flow Cytometric Analysis
Cell Fixation/Permeabilization Kits
Immunofluorescent Staining of Intracellular Cytokines for Flow Cytometric Analysis
Annexin V Staining Protocol
APO-BRDU™ Procedure
APO-DIRECT™ Procedure
Detection of BrdU Incorporation in DNA Synthesizing Cells
Staining Procedure for Flow Cytometric Detection of Human Cyclins
Indirect Immunofluorescence Staining of Human Platelets
Immunofluorescence Staining of Human Cells by Lysed Whole Blood Method
Immunofluorescent Staining of Mouse and Rat Leukocytes
The Uses of Fc Block in Immunophenotyping of Mouse or Rat Leukocytes
ELISA/ELISPOT
Cytokine ELISA
Mouse IgE ELISA Protocol
Immunohistochemistry
Preparation and Staining of Frozen Tissue Sections
Preparation and Staining of Paraffin Sections
Microwave citrate Pretreatment of Paraffin Sections
Immunohistochemistry / Tissue Section Staining
Cell Staining for Immunofluorescence Microscopy
Western Blotting
Western Blotting with Alkaline Phosphatase Conjugates
Western Blotting with Biotinylated Antibodies
Western Blotting with Horseradish Peroxidase Conjugates
Western Blotting with Monoclonal Antibodies
Western Blotting with Rabbit Polyclonal Antibodies
Preparation of Brain Membrane Fractions for Western Blot Analysis
Immunoprecipitation
Immunopurification of Tyrosine Phosphorylated Proteins
Immunoprecipitation with Antibody:Agarose Conjugates
Immunoprecipitation with anti-Phosphotyrosine: Biotin Conjugates
Immunoprecipitation With Soluble Antibodies
Immunoprecipitation of 35S Labeled Cells
RNAse Protection Assay (RPA)
RNase Protection Assay
Total RNA Isolation
Protein Kinase Assay (PKA)
Protein Kinase Assay with an Immunocomplex
Protein Kinase Assay after Denaturation and Renaturation




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