CD41-PE, P2, 2 mL, ASR

Protocol tips

Upstream tips	Protocol tips	Downstream tips
For platelet staining, 100 μL of PRP was aliquoted into 12 × 75 mm round bottom tubes	The samples were incubated in a dark environment for 30 min at room temperature. After incubation, 500 μL of PBS was added to each tube and the samples were analysed on the BD FACSAria IIu cell sorter located in the CAF Fluorescence Microscopy Unit, Stellenbosch University. For each sample, a minimum of 30,000 events were acquired and all signal were gated.

Upstream tips
For platelet staining, 100 μL of PRP was aliquoted into 12 × 75 mm round bottom tubes
Protocol tips
The samples were incubated in a dark environment for 30 min at room temperature. After incubation, 500 μL of PBS was added to each tube and the samples were analysed on the BD FACSAria IIu cell sorter located in the CAF Fluorescence Microscopy Unit, Stellenbosch University. For each sample, a minimum of 30,000 events were acquired and all signal were gated.

Publication protocol

For platelet staining, 100 μL of PRP was aliquoted into 12 × 75 mm round bottom tubes (BD Biosciences, 352063). Thereafter, 20 μL of PAC-1 FITC (BD Biosciences, 340507) and 20 μL of CD41 PE (Beckman Coulter, IM1416U) stored in a phosphate buffered saline storage solution with gelatin and 0.1% sodium azide, were added to the PRP and gently mixed by pipetting. The samples were incubated in a dark environment for 30 min at room temperature. After incubation, 500 μL of PBS was added to each tube and the samples were analysed on the BD FACSAria IIu cell sorter located in the CAF Fluorescence Microscopy Unit, Stellenbosch University. For each sample, a minimum of 30,000 events were acquired and all signal were gated. The addition of prostaglandin (which is usually added to prevent platelet activation during a second step where a platelet pellet is needed), was omitted since PRP was obtained by centrifuging WB only once, at a very low relative centrifugal force (150×g). For compensation, single stained platelets were used to determine optimal voltages and Anti-Mouse Ig compensation beads (BD Biosciences, 552843) were used to determine the compensation matrix. To ensure the consistency and reproducibility of the data, application settings were set and applied to the experiment and eight peak beads were used as a secondary measure. Platelets were identified and gated using SSC vs CD41-PE dot plot and the PAC-1 positive and negative cells were identified from this population. All analyses were performed using FlowJo v10.4.1, and data were exported to Microsoft Excel for further analysis.

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Manufacturer protocol

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