Pe conjugated anti cd31

PE-conjugated anti-CD31 was from eBioscience (San Diego, CA, USA). APC-conjugated anti-CD42b was purchased from BD Bioscience (Erembodegem, Belgium). FITC-conjugated anti-CD62E (E-Selectin) was obtained from Santa Cruz Biotech (Santa Cruz, CA, USA). FITC-conjugated Annexin V was from ImmunoStep (Salamanca, Spain). Recombinant fibronectin, thrombin and other chemicals were provided by Sigma-Aldrich (St Louis, MO, USA).

Immunofluorescence staining was performed on cultured LMCs at 24 h after isolation using PE-conjugated anti-CD73 (BD Bioscience, USA, #127205, 1/150), PE-conjugated anti-CD31 (eBioscience, USA, #12-0311-82, 1/150), FITC-conjugated anti-CD68 (BioLegend, #137005, 1/300), and PE-conjugated anti-CD34 (BD Bioscience, #551387, 1/75). Briefly, the cells were washed with PBS and 4% of cold paraformaldehyde was added for 20 min. Then, 5% of goat serum was added for 60 min at RT for blocking, and then, remove without washing. The cells were incubated with the antibodies diluted in BSA/PBS 0.1% for 1–2 h at RT after which the wells were washed with PBS–Tween %0.1, 3 × 5 min. The cells were kept in PBS , and imaging was performed using Nikon Eclipse Ti (Nikon). The ratio of positive cells for each marker was determined by inspecting 100 LMCs.