Flow cytometric analysis of mouse platelet surface glycoproteins

-Preparation of diluted or washed whole blood

-Preparation of washed mouse platelets

-Single-color analysis of platelet surface glycoprotein expression

-Two-color analysis of platelet activation

Immunohistochemistry (with acetone-fixed frozen sections)

Immunoprecipitation

Immunoblot (Western Blot Analysis)

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Emfret Flow cytometric analysis of mouse platelet surface glycoproteins

Buffers and reagents

Tris buffered saline/Heparin (TBS/Hep): 20 mM Tris/HCl; 137 mM NaCl; pH 7.3; containing 20 U/ml Heparin.

Phosphate buffered saline (PBS): 137 mM NaCl; 2.7 mM KCl; 1.5 mM KH2PO4; 8 mM Na2HPO4; pH 7.14.

Tyrode’s Buffer (modified): 134 mM NaCl; 0.34 mM Na2HPO4; 2.9 mM KCl; 12 mM NaHCO3; 20 mM Hepes; pH 7.0; 5 mM glucose; 0.35% (w/v) bovine serum albumin

Apyrase: 10 U/ml stock in H2Obidest (stored at -20°C)

Prostacyclin (Prostaglandin 2, PGI2): 1 mM stock in H2Obidest (stored at -20°C)

CaCl2: 1 M stock in H2Obidest

Preparation of diluted or washed whole blood

1. Take 50 µl blood with a heparinized glass capillary (e.g. ringcap) from the retro-orbital plexus into 1.5 ml tubes containing 200 µl of TBS/Heparin (20 U/ml).
2. Dilute in 1 ml of Tyrode´s buffer and use for flow cytometric analysis.
3. To prepare washed blood, centrifuge the diluted blood at 900 x g for 5 min, remove the supernatant and resuspend the pellet in 1.25 ml Tyrode´s buffer.
4. Add 1 mM CaCl2 directly before the start of the experiment.
   

Note: For thrombin-induced platelet activation, plasma has to be removed to prevent anti-thrombin activity

Preparation of washed mouse platelets

The preparation of washed platelets is a more time consuming method, that requires a larger amount of blood, but yields a platelet preparation that can be used for several hours.

1. Collect 0.5 - 1 ml blood with 1.5 cm glass capillaries from the retro-orbital plexus into a 1.5 ml tube containing 200 µl of TBS/Heparin (20 U/ml).
2. Centrifuge the sample for 5 min at 500 x g and transfer the platelet rich plasma (PRP) into a new tube. For best recovery of platelets, take the complete white phase including some red blood cells.
3. Centrifuge the platelet suspension for 8 min at 300 x g, and transfer the PRP without any red blood cells into a new tube.
4. Add 0.5 µM prostacyclin (PGI2) and centrifuge at 1300 x g for 5 min.
5. Resuspend the platelet pellet in 1 ml Tyrode’s buffer, add 0.02 U/ml apyrase and 0.5 µM prostacyclin, incubate for 5 min at 37°C, and centrifuge for 5 min at 1300 x g.
6. Repeat step 5 and resuspend the platelet pellet in 0.5 ml Tyrode’s buffer, add 0.02 U/ml of apyrase and incubate for 30 min at 37°C.

Single color analysis of platelet surface glycoproteins

1. Combine 5 µl of specific or negative control antibodies and 25 µl diluted whole or washed blood in the assay tube and vortex mix for 1-2 seconds.
2. Incubate for 15 min at room temperature.
3. Stop reaction by addition of 400 µl PBS and analyze within 30 min.

Two color analysis of platelet activation

1. Add 5 µl specific or negative control antibodies to the assay tube together with 5 µl of a pan-platelet marker.
2. At this point, any additional reagents (e.g. inhibitors) are added in small volumes (< 5 µl).
3. Add 26 µl diluted whole or washed blood or washed platelets (1 million) and vortex mix for 1-2 seconds.
4. Incubate for 15 min at room temperature. Stop reaction by addition of 400 µl PBS and analyze within 30 min.