Platelet-poor plasma (PPP) was prepared from the remaining sample after removal of PRP by centrifugation at 10.000×g for 10 minutes. PRP was pre-incubated with drug or vehicle. Half-area 96-well microtiter plates (Greiner Bio-One, Stonehouse, Gloucestershire,UK) were coated with 5 µL PBS with or without the addition of collagen (COLTest, Roche Diagnostics, Mannheim, Germany) as previously described [29] . The final concentrations of collagen were: 30.0, 12.8, 6.4, 3.2, 1.1, 0.36 and 0.12 µg/mL. Then, 45 µL PRP was added to each well. As reference samples, PRP and PPP were added to wells coated with only PBS. Next, microtiter plates were shaken at 900 rpm for 10 min at 37°C on the Victor X5 (Perkin Elmer, Turku, Finland) with subsequent readings of absorbance and reporting as optical density (OD). Percent of platelet aggregation was calculated using the formula [29] : 100% × (ODPRP -ODsample) / (ODPRP -ODPPP)
Half area 96 well microtiter plates
Manufactured by Greiner
Sourced in United Kingdom
Half-area 96-well microtiter plates are a type of laboratory equipment designed for various experimental applications. These plates feature a reduced well volume compared to standard 96-well plates, allowing for efficient use of sample and reagent volumes. The half-area format provides a compact and space-saving solution for researchers and scientists working with limited sample quantities or aiming to minimize reagent consumption.
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2 protocols using half area 96 well microtiter plates
1
Collagen-Induced Platelet Aggregation Assay
2
Whole Blood Platelet Aggregation Assay
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Platelet aggregation was performed in whole blood as previously described.19 (link) Briefly, in half-area 96-well microtiter plates (Greiner Bio-One), whole blood anticoagulated with lepirudin was incubated with PBS, arachidonic acid (0.05-0.5 mM; Sigma), the Horm collagen (0.1-10 μg/mL; Takeda), PAR4-amide, (50-100 μM), or U46619 (0.1-10 μM) under mixing conditions (1200 revolutions per minute, Bioshake iQ, Qinstruments). Samples were subsequently diluted (1:20 acid citrate dextrose) and analyzed by flow cytometry (Novocyte) to identify and determine proportions of the single-platelet population.
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