Product Description
FITC-Conjugated anti-human P-selectin (CD62) chicken antibodies
Cat. # 2874
FITC-conjugated anti-human P-selectin (CD62) chicken antibodies are intended to be used for flow cytometric measurement of platelet activation, spontaneous or induced, utilising exposure of P-selectin (CD62P) as marker of platelet activation.
Reagent for up to 100 test
For Research Use Only
Intended Use
FITC-conjugated anti-human CD62P antibodies are intended to be used for quantification of platelet activation, as measured by platelet exposure of P-selectin using flow cytometry.
1. Assay Principle
Platelet rich plasma (PRP) from patients or healthy control subjects is incubated with buffer and FITC-conjugated anti-human CD62P. The FITC-anti-CD62P antibody will bind to activated platelets by binding to exposed P-selectin. The amount of fluorescence bound to the platelets is used to determine the proportion of activated platelets in the PRP or whole blood.
The degree of activation is determined both for non-stimulated platelets and platelets stimulated with different concentrations of ADP or other agonists.
2. Reagents Provided
FITC-conjugated anti-CD62P: 1 vial of 1 mL ready to use chicken- anti-CD62P antibodies, conjugated with fluorescein isothiocyanate (FITC).
The solvent is 0.02 M sodium phosphate, 0.15 M NaCl and 0.02 % sodium azide, pH 7.2.
The conjugated antibody should be stored dark at +2-6°C.
3. Warning
The FITC-conjugated antibody solution contains sodium azide which may react with lead and copper plumbing to form highly explosive metal azides. Materials discarded into the sink should be flushed with a large volume of water to prevent azide build-up.
4. Reagents and Equipment Required but not Provided
HEPES-buffer: 20 mmol/L HEPES, 137 mmol/L NaCl, 2.7 mmol/L KCl, 1 mmol/L MgCl2, 5.6 mmol/L glucose, 1 g/L bovine serum albumin, pH 7.40.
ADP: 1.7 and 8.5 µmol/L in HEPES-buffer
Plastic tubes, 2.5 / 5 mL capacity
Flow cytometer
5. Sample Collection
Nine volumes of venous blood are collected in 1 volume of 0.1 M trisodium citrate. Prepare platelet rich plasma (PRP) by centrifugation at 140 x g for 10 minutes at room temperature. For determination of spontaneous platelet activation, the sample must be assayed immediately. For determination of activation after stimulation with ADP, the samples should be kept at room temperature and analysed between 0.5 and 4 h after sampling.
6. Assay Procedure
6.1 Preparation of Samples
Dissolve ADP in HEPES-buffer to final concentrations of 0, 1.7 and 8.5 µM respectively. For each sample, 20 µL of the respective solutions is needed.
Add to three plastic tubes:
230 µL HEPES-buffer
10 µL FITC-conjugated anti-CD62P
20 µL PRP
Incubate for 10 minutes at RT
Add 20 µL of the respective ADP solutions (0, 1.7 and 8.5 µM) to the three tubes.
Incubate for exactly 10 minutes at RT
Add 2 mL ice cold HEPES-buffer
Analyse on a flow cytometer according to the instructions of the manufacturer. The analytical markers in the fluorescence channel are used to divide a negative control sample, i.e. not activated platelets or using a FITC-conjugated control antibody, into two fractions containing 98-99 % of the platelets and 1-2 % of the brightest representatives of the platelet population. Platelets with fluorescence greater than the marker are identified as positive events.
The fraction (in %) activated platelets at different ADP concentrations are calculated and compared to the degree of activation with and without stimulation for healthy normal subjects.
7. Limitations
Vigorous stirring of platelets must be avoided.
Ver 030309