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BiochemicalsProteins and Peptides
Proteins and peptidesAnti-Ly6g antibody [1A8] - mouse IgG2c (Chimeric)
Low endotoxin, Azide free.
Our first-to-market chimera with mouse IgG2c backbone, this functional antibody specifically depletes neutrophils in vivo for up to 72h.
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To prevent the activation of platelets during the procedure, strong mechanical forces (e.g. fast pipetting, vigorous shaking) should be avoided. In addition, the platelet inhibitors indicated in the protocol can be used, but other inhibitors exist as well. It is the researcher's choice to select inhibitors that are most suitable for their studies and experimental goals.
Examples of applications for isolated platelets are: western blotting, flow cytometry, ELISA, and immunocytochemistry/ immunofluorescence, to investigate cellular and signaling processes within the platelets. Platelet aggregation and adhesion upon stimulation with various agents can be studied as well as the release of granule components upon activation.
Unless platelets are used in tissue culture experiments, the reagents do not need to be sterile. Store and use all buffers at 4°C, unless indicated otherwise.
Notes on blood collection
The first step of this procedure includes obtaining the whole blood and preparing platelet-rich plasma (PRP) by centrifugation. The centrifugation conditions can vary widely (e.g. from 800 x g for 5 min to 100 x g for 20 min), but give consistently good separations.
A phlebotomist should draw blood into a Becton Dicinson Vacutainer® (containing ACD, yellow cap) or into a plastic syringe containing 1/10 volume of CPD buffer. The volume of blood needed depends on the experiment. A volume of 40–45 ml blood results in average 1–3 x 109 platelets. The platelet number can vary depending on the individual the blood is drawn from.
Mix gently during and after blood collection by slowly inverting the Vacutainer® or syringe.
Spin at 200 x g for 20 min at room temperature (with no brake applied).
After the spin, three distinct layers can be observed:
Transfer two thirds of the PRP from the blood fractionation into a new plastic tube using a transfer pipette (wide orifice).
Add HEP buffer at a 1:1 ratio (v/v). Include prostaglandin E1 (PGE1, 1 µM final concentration) to prevent platelet activation.
Mix very gently by inverting the tube slowly.
Spin at 100 x g for 15–20 min at room temperature (with no brake applied) to pellet contaminating red and white blood cells.
Transfer the supernatant into new plastic tube using a transfer pipette (wide orifice).
Pellet platelets by centrifugation at 800 x g for 15–20 min at room temperature (with no brake applied).
Rinse the platelet pellet with platelet wash buffer (without resuspension in order to avoid unnecessary platelet activation) by gently adding wash buffer and removing it slowly with a pipette.
Carefully and slowly resuspend the pellet in Tyrode's buffer containing 5 mM glucose and freshly added BSA (3 mg/ml) using a transfer pipette (wide orifice).
If the platelets need to be activated in subsequent experiments, omit the addition of PGE1 and apyrase at this step.
For preparation of a lysate of quiescent platelets, add instead an ice-cold 1:1 mixture of Tyrode's buffer and 2X platelet lysis buffer, including protease inhibitors.
Count the platelets (e.g. by using a hemocytometer, semi-automated Coulter-type counters or an electronic particle analyzer).
Adjust the platelet concentration as needed with Tyrode's buffer.
A common concentration used for various platelet studies (e.g. platelet activation, aggregation and adhesion assays, western blotting and immunocytochemistry/immunofluorescence) is 1–3 x 108 platelets/mL.