Emfret

德國(guó)Emfret公司專注心血管和血液系統(tǒng)研究用抗體研發(fā)和生產(chǎn)，供應(yīng)的抗體包括**血液中血小板、流式細(xì)胞術(shù)鑒定分析小鼠血小板表面糖蛋白，Emfret公司的抗體不僅適用于流失分析、WB檢測(cè)，還可用于**組化/**熒光及**共沉淀檢測(cè)。

Emfret Technical Protocols

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)

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

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).

Dilute in 1 ml of Tyrode′s buffer and use for flow cytometric analysis.

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.

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.

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).

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.

Centrifuge the platelet suspension for 8 min at 300 x g, and transfer the PRP without any red blood cells into a new tube.

Add 0.5 μM prostacyclin (PGI2) and centrifuge at 1300 x g for 5 min.

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.

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

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.

Incubate for 15 min at room temperature.

Stop reaction by addition of 400 μl PBS and analyze within 30 min.

Samples were incubated with FITC-conjugated control IgG (black line), anti-GPVI, or anti-GPV as described. Platelets were gated by forward (FSC) / side scatter (SSC) characteristics. Fluorescence 1 (FL1) intensity of the gated (R1) platelets is shown in separate histograms. RBC: red blood cells.

Two color analysis of platelet activation

Add 5 μl specific or negative control antibodies to the assay tube together with 5 μl of a pan-platelet marker.

At this point, any additional reagents (e.g. inhibitors) are added in small volumes (< 5 μl).

Add 26 μl diluted whole or washed blood or washed platelets (1 million) and vortex mix for 1-2 seconds.

Incubate for 15 min at room temperature. Stop reaction by addition of 400 μl PBS and analyze within 30 min.