Flow cytometry for assessment of cell surface and MP expression from cancer cells lines and HUVEC was performed on a FACSCalibur (Becton Dickinson, Oxford, UK). For assessment of cell free media was obtained by centrifugation at 400g for 5 minutes to pellet cells. 25µl of this media was then incubated with 5µl of antibody (CD31:FITC, CD54:FITC, CD105:FITC, CD106:FITC or CD142:FITC) (Bio-Rad, Hemel Hempstead, UK) at room temperature in the dark for 30 minutes, when counting beads (Accucheck, Thermo-Fisher) and 150µl filtered PBS were added prior to assessment by flow cytometry using ISTH guidelines for enumeration of MP.
For assessment of cancer or HUVEC cells, 5µg of antihuman antibodies (CD31:FITC, CD54:FITC, CD105:FITC, CD106:FITC or CD142:FITC, Bio-Rad, Hemel Hempstead, UK) or an isotype matched negative control (IgG1:FITC, Bio-Rad) was then added to 50µl of cells (2x10 5 ) and incubated for 30 minutes, at room temperature in the dark. Cells were then washed in PBS and centrifuged. The pellet was then resuspended in 200µl filtered PBS and assessed by flow cytometry.
For assessment of cancer or HUVEC cells, 5µg of antihuman antibodies (CD31:FITC, CD54:FITC, CD105:FITC, CD106:FITC or CD142:FITC, Bio-Rad, Hemel Hempstead, UK) or an isotype matched negative control (IgG1:FITC, Bio-Rad) was then added to 50µl of cells (2x10 5 ) and incubated for 30 minutes, at room temperature in the dark. Cells were then washed in PBS and centrifuged. The pellet was then resuspended in 200µl filtered PBS and assessed by flow cytometry.