PE-Cy™7 Mouse Anti-Human CD41a

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	To measure microparticles, plasma aliquots were centrifuged for 2 min at 10,000xg to pellet residual cells and debris. The supernatant was collected and then centrifuged for 45 min at 17,000xg. The resulting microparticle pellet was resuspended in PBS containing 1% FCS and 2.5mM Ca+2 and incubated with Fc block for 10 min at 4°C.	After 30 min at room temperature, 25,000 volumetric counting beads were added and 10,000 events collected on an LSR II flow cytometer.

Protocol tips
To measure microparticles, plasma aliquots were centrifuged for 2 min at 10,000xg to pellet residual cells and debris. The supernatant was collected and then centrifuged for 45 min at 17,000xg. The resulting microparticle pellet was resuspended in PBS containing 1% FCS and 2.5mM Ca+2 and incubated with Fc block for 10 min at 4°C.
Downstream tips
After 30 min at room temperature, 25,000 volumetric counting beads were added and 10,000 events collected on an LSR II flow cytometer.

Publication protocol

To measure microparticles, plasma aliquots were centrifuged for 2 min at 10,000xg to pellet residual cells and debris. The supernatant was collected and then centrifuged for 45 min at 17,000xg. The resulting microparticle pellet was resuspended in PBS containing 1% FCS and 2.5mM Ca+2 and incubated with Fc block for 10 min at 4°C. Then a cocktail of fluorescently conjugated antibodies including, Pacific blue-Annexin V (Life Technologies), APC-anti-CD34 (Becton Dickinson), PECy7-anti-CD41 (Becton Dickinson), PE-anti-CD31 (eBioscience), PECy5.5-anti-CD62E (Becton Dickinson), FITC-anti-EphB4 (R&D Systems), and APC-anti CD143 (Biolegend) was added. In addition, one antibody (anti-Ephrin B2; Santa Cruz) was labeled in the laboratory (Zenon Alexa 488 labeling kit; Life Technologies) and also added to the staining cocktail. After 30 min at room temperature, 25,000 volumetric counting beads were added and 10,000 events collected on an LSR II flow cytometer. An identical sample with no antibodies was used as a gating control. Microparticle numbers were quantified in gated populations <1μm in size and positive for Annexin V staining using the FlowJo software package and normalized to the number of counting beads (volume) collected. Specific populations were defined by phenotype as documented in Table 2 and illustrated in Supplemental Figure I in the Online Data Supplement.

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