CD61-FITC, SZ21, 2 mL, ASR

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Blood was centrifuged at 800 ×g for 20 minutes at room temperature to obtain platelet-rich plasma. Then, 1.5 mL supernatant was transferred into a new test tube and centrifuged at 1,500 ×g for further 20 minutes to obtain platelet-poor plasma (PPP). Afterward, 1 mL PPP was further centrifuged at 1,500 ×g for 20 minutes in a new polystyrene tube to obtain cell-free plasma. The top 500 µL of cell-free plasma was transferred into an Eppendorf tube and pelleted at 18,000 ×g for 10 minutes. The supernatant was carefully removed leaving 25 µL of MP-rich plasma at the bottom of the Eppendorf tube. MPs were suspended with gentle vortexing for 20 seconds in 1.0 mL Apo-binding buffer

Protocol tips

Blood was centrifuged at 800 ×g for 20 minutes at room temperature to obtain platelet-rich plasma. Then, 1.5 mL supernatant was transferred into a new test tube and centrifuged at 1,500 ×g for further 20 minutes to obtain platelet-poor plasma (PPP). Afterward, 1 mL PPP was further centrifuged at 1,500 ×g for 20 minutes in a new polystyrene tube to obtain cell-free plasma. The top 500 µL of cell-free plasma was transferred into an Eppendorf tube and pelleted at 18,000 ×g for 10 minutes. The supernatant was carefully removed leaving 25 µL of MP-rich plasma at the bottom of the Eppendorf tube. MPs were suspended with gentle vortexing for 20 seconds in 1.0 mL Apo-binding buffer

Publication protocol

"Currently, there is no standardized method for MP analysis. Because of this, and to avoid false-positive MP results, we developed a method for MP isolation as follows.

Blood was centrifuged at 800 ×g for 20 minutes at room temperature to obtain platelet-rich plasma. Then, 1.5 mL supernatant was transferred into a new test tube and centrifuged at 1,500 ×g for further 20 minutes to obtain platelet-poor plasma (PPP). Afterward, 1 mL PPP was further centrifuged at 1,500 ×g for 20 minutes in a new polystyrene tube to obtain cell-free plasma. The top 500 µL of cell-free plasma was transferred into an Eppendorf tube and pelleted at 18,000 ×g for 10 minutes. The supernatant was carefully removed leaving 25 µL of MP-rich plasma at the bottom of the Eppendorf tube. MPs were suspended with gentle vortexing for 20 seconds in 1.0 mL Apo-binding buffer (10 mmol/L HEPES-4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid [Sigma-Al-drich Co., St Louis, MO, USA], 5 mmol/L KCl, 1 mmol/L MgCl2, 136 mmol/L NaCl, pH =7.4) without CaCl2.

In this study, only Annexin V+ MPs were analyzed. We selected EMPs (CD31+, CD62E+) and platelet-derived (CD61+, CD41+, CD42a+, PAC1+), red-blood-cell-derived (GlyA+) and leukocyte-derived (CD45+, CD13+, CD14+, CD 56+) MPs. The selected CD markers, their cellular origin, the fluorescent dye used for labeling and the manufacturer’s specification for our MP measurements are summarized in Table 1. The individual antibody cocktail tubes are listed in Table S1."

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