Platelet-rich plasma (PRP) was isolated, and 25 µl of PRP was incubated with platelet activation markers PAC-1-FITC (BD, catalog number 340507) and CD62P)-APC (BD, catalog number 550888) for 20 min at room temperature. The platelets were washed with phosphate-buffered saline (PBS), resuspended in PBS, and analyzed in Attune NxT flow cytometer (Thermo Fisher Scientific, Singapore). The gating strategy for measuring the percent of platelet activation is described in Supplementary Figure S3
Cd62p apc
Manufactured by BD
Sourced in United States
CD62P)-APC is a fluorescently labeled antibody that binds to the CD62P antigen, also known as P-selectin. It is commonly used in flow cytometry applications to identify and quantify cells expressing the CD62P antigen.
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Lab products found in correlation
Pac 1 fitc, by BD (1 mentions)
Sodium azide, by Thermo Fisher Scientific (1 mentions)
Igg1 pe, by BD (1 mentions)
Igg1 apc, by BD (1 mentions)
Vegf a, by Merck Group (1 mentions)
Viability dye 7 aad, by BD (1 mentions)
Igg1 fitc, by BD (1 mentions)
Accuri c6 flow cytometer, by BD (1 mentions)
Icam1 pe, by BD (1 mentions)
Vcam 1 fitc, by BD (1 mentions)
Jama pe, by BD (1 mentions)
Fcs express 4 flow cytometry research edition software, by De Novo Software (1 mentions)
Rpmi media, by Thermo Fisher Scientific (1 mentions)
Cd44 pe cy7, by BD (1 mentions)
Sodium azide, by Merck Group (1 mentions)
Tnf α, by R&D Systems (1 mentions)
Cflow plus software, by BD (1 mentions)
Cd14 pe cy7, by BioLegend (1 mentions)
Cd19 percp cy5, by BD (1 mentions)
Cd3 fitc, by BioLegend (1 mentions)
3 protocols using cd62p apc
1
Platelet Activation Assay Using Flow Cytometry
2
Immunophenotyping of Cell Adhesion Molecules
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Cells were harvested with 0.01 M EDTA/PBS, blocked with human IgG (0.1 mg/ml in RPMI media (Life Technologies), 10% fetal bovine serum (FBS, Hyclone, Logan, UT, USA), 0.1% sodium azide (Thermo Fisher Scientific, Waltham, MA, USA) for 10 min on ice prior to staining with an antibody master mix (CD44-PECy7, CD62P-APC, CD62E-APC, ICAM1-PE, ICAM2-APC, VCAM1-FITC, JAMA-PE (all BD), JAMC-VioBright FITC (Miltenyi Biotec, Bergisch Gladbach, Germany)) or the respective isotype controls (IgG2-PECy7, IgG1-APC, IgG1-PE, IgG1-FITC (all BD Biosciences, Franklin Lakes, NJ, USA), REA Control VioBright FITC (Miltenyi Biotec)) with viability dye 7-AAD (BD) in RF10 at manufacturer’s recommended concentrations for 30 min, 4°C in the dark. Cells were washed, fixed with FACS fix (PBS with 1% paraformaldehyde, 20 g/L glucose and 5 mM sodium azide (all Sigma-Aldrich, St Louis, MO, USA)) and analyzed on the C6 Accuri™ flow cytometer (BD) using the CFlow® Plus software (BD Biosciences) and FCS Express 4 Flow Cytometry: Research Edition software (De Novo, Pasadena, CA, USA).
In similar experiments, ECFCs and C32 melanoma cells were treated without or with 50 ng/ml VEGF-A (Sigma) for 72 h with the final 24 h including (or not) 10 ng/ml TNFα (R&D Systems). Cells were then assessed for cell surface expression of ICAM-1 and cell viability as detailed above.
In similar experiments, ECFCs and C32 melanoma cells were treated without or with 50 ng/ml VEGF-A (Sigma) for 72 h with the final 24 h including (or not) 10 ng/ml TNFα (R&D Systems). Cells were then assessed for cell surface expression of ICAM-1 and cell viability as detailed above.
3
Flow Cytometric Analysis of Extracellular Vesicles
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Extracellular vesicles phenotyping was performed using a flow cytometer (FC) as previously described [15 , 16 ]. Briefly, 20 μl of the supernatant of each RBC product was stained with the following linage‐specific monoclonal antibodies to identify the cell of origin of EVs: CD41a‐PerCP‐Cy5.5, CD142‐APC, CD66b‐PE, CD144‐BV421, CD235a‐FITC, CD3‐FITC, and CD14‐PE‐Cy7 (Biolegend, San Diego, CA, USA), and CD16‐ECD, CD19‐PerCP‐Cy5.5 and CD62P‐APC (BD Biosciences, Chatham, NJ, USA). Stained samples were run through an LSRII flow cytometer (BD Biosciences); sufficient events were collected to provide approximately ≥5·000 gated EV events. An AbC Anti‐Mouse Bead Kit (Life Technologies, Carslbad, CA, USA) was used to set the compensation along with the single‐stained compensation control. Small size beads, ranging from 0.2 to 1 μm (Megmix‐Plus SSC beads, Biocytex, Marseille, France), were used to generate the EV gate and to further classify them based on their size (only EVs ≤ 1.0 µm in diameter were analysed). BD TruCOUNT tubes (BD Biosciences) were used to obtain the absolute number of EVs/μL. Data were analysed using FlowJo v10.
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