CytoFlex Flow Cytometer Application Notes

Working solutions

1 x PBS sterilized through 0.2 µ m filter

CD62P-PE antibody

Prepare:

• 1:32 dilution of antibody from stock:

• 2 µ L Ab + 62 µ L 1xPBS = 64 µ L of 1:32 dilution anti-CD62P-PE antibody

ADP

• Prepare 1 mM stock according to manufacturer’s instructions then make a 100 µ M working solution.

• 3 µ L 1 mM ADP + 27 µ L 1xPBS = 30 µ L of 100 µ M ADP.

Procedure

A. Blood collection

Collect blood aseptically by venipuncture using a 20-gauge needle. Collect the first ~2 mL of blood into any vacutainer; discard this sample as it contains activated platelets. Release tourniquet and collect an additional 3.5 mL of blood into sodium citrate tube; this sample will be used for staining.

B. Activation and Staining of Platelets

1. Prepare the working solutions (see above). Keep at room temperature.

2. Label polypropylene tubes for flow cytometry (12x75 mm) as follows in duplicate:

1. No Ab, no ADP

2. CD62P no ADP

3. CD62P + ADP

3. Add reagents according to Table 1 to the appropriate tubes. Use a fresh tip for each addition and swirl tubes gently to mix.

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