File Name : supplementary flow cytometry.tif

Caption : supplementary figure 1. comparative assessment of the α1, α2 and β1 integrin profile of haecs versus baecs. the expression of integrins α1, α2 and β1 was assessed by flow cytometry using the agilent 2100 bioanalyzer microfluidic system.  briefly, 200,000 cells were suspended in 1.7 ml conical tubes (on ice) and stained with 2 µm calcein am (life technologies) in staining buffer (1% bsa in dpbs) for 30 min.  following incubation, samples were centrifuged at 600xg at 4 °c and rinsed twice with staining buffer.  the cell pellet was then incubated for 30 min in 100 µl of appropriate primary antibody or igg control (α1: clone fb12, chemicon; α2: clone p1e6, chemicon; β1: clone 6s6, chemicon) dissolved in staining buffer at 10 µg/ml.  thereafter, the cells were centrifuged again and the pellet rinsed twice with 1 ml of staining buffer and the secondary antibody (alexafluor 647 conjugated donkey igg, jackson immunoresearch) was applied at 10 µg/ml in staining buffer for 30 min.  finally, the cell pellet was rinsed twice with 1 ml staining buffer and re-suspended at 1 x 106 cells/ml in loading buffer (agilent) and loaded in to the microfluidic chip (agilent) according to the manufacturer’s instructions.