FITC-Conjugated anti-human complement factor 3 (C3)
chicken antibodies
Cat. # 2875
FITC-conjugated anti-human C3 chicken antibodies
are intended to be used for flow cytometric
measurement of platelet activation, spontaneous or induced, utilising
exposure of platelet bound C3 as marker of platelet activation.
Price: 380 Euro
Order now at mail: Info@diapensia.se
Reagent for up to 100 test
For Research Use Only
Intended Use
FITC-conjugated anti-human C3 antibodies are
intended to be used for quantification of platelet activation, as measured by
platelet exposure of platelet bound C3 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 C3. The FITC-anti-C3 antibody will bind to activated platelets by
binding to platelet bound C3. The amount of fluorescence bound to the
platelets is used to determine the proportion of platelets binding C3 in the PRP or whole blood.
The degree of activation is determined both for non-stimulated platelets and
platelets stimulated with different concentrations of immune complexes or
other agonists.
2.Reagents Provided
FITC-conjugated anti-C3: 1 vial of 1 mL ready to use chicken- anti-C3 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.
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
Add to three plastic tubes:
230 µL HEPES-buffer
10 µL FITC-conjugated
anti-C3
20 µL PRP (heparin or hirudin as anticoagulant is recommended)
Incubate for 10 minutes at RT
Add 20 µL of agonist to two 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
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