Fitc conjugated cd31

The number of cells in single-cell suspension was estimated using a Scepter™ automated cell counter (Millipore-Sigma, Burlington, MA, USA) and a total of 0.5×10⁶ cells/sample were resuspended in 200 μl of PBS in 96-well plate. Cells were incubated in the dark with 2 μl/1 × 10⁶ cells of CD16/32 antibody (Fc block; BD Biosciences, Mississauga, ON, Canada) for 15 min at RT. Following blocking, cells were centrifuged and resulting pellets were resuspended in 1:100 mixture of panel of antibodies: FITC-conjugated CD31 (BD Biosciences, Mississauga, ON, Canada), AF647-conjugated CD45 (Southern Biotech, Birmingham, AL, USA) and Pe/Cy7-conjugated CD326 (EpCAM; Thermofischer Scientific, Burlington, ON, Canada). Cells were incubated with antibodies at RT for 30 min in dark. Following staining, cells were pelleted by centrifugation and washed 3× with FACS buffer (5% (v/v) FBS and 1 mM EDTA in 1×DPBS). All samples were fixed by 4% (w/v) PFA prior to analysis. Flow cytometry was performed using a BD LSR Fortessa (Beckton Dickinson Biosciences, Franklin Lakes, NJ, USA) at the Ottawa Hospital Research Institute (OHRI) core facility. Sample compensation was performed using BD FACSDIVA software and data analysis were performed with FlowJo v10 software (FlowJo LLC, Ashland, OR, USA) (Supplementary Fig. 10).