Enzymatic free cell dissociation buffer

RCMVECs were washed three times with Dulbecco’s phosphate-buffered saline (DPBS, Invitrogen), lifted using Enzymatic Free Cell Dissociation Buffer (Millipore) for 15 minutes at 37°C, manually disrupted if necessary, collected, centrifuged at 100×g for 5 minutes, and washed twice with DPBS. RCMVECs were resuspended in 1 mL of the appropriate media per treatment group, counted on a Cell Countess, and diluted accordingly. 20,000 RMVECs were added accordingly to each chamber in 1 mL of media on a four-chamber slide (Nunc Lab-Tek). Chambers were coated with 250 μL of Growth Factor Reduced Matrigel (BD Biosciences) under chilled conditions followed by solidification at room temperature prior to RCMVEC addition. After 24 and 48 hours incubation at 37°C on the Matrigel, 4× and 10× magnification images were taken of the RCMVEC tube formation on a Nikon TS-100 Microscope with Flex camera (Nikon).

Tube formation assay was performed as in previous studies[3 (link)], with the exception of the slide format used. SS-AT₁WT and SS-AT₁KO rat cardiac microvascular endothelial cells (RMVECs) (Cell Biologics) grown according to company protocol were brought to 70–90% confluency, washed twice with DPBS, and lifted using Enzymatic Free Cell Dissociation Buffer (Millipore) with gentle agitation for 30 minutes at 37°C. RMVECs were then centrifuged at 300 x g for 5 minutes, washed twice with DPBS, resuspended in 1 mL MCDB131 basal media plus 2% FBS, and counted using the cell Countess system (Invitrogen). RMVECs were diluted for the addition of 1,250 cells in 50uL of media per well of u-Angiogenesis slides (iBidi) coated in 11 uL of Geltrex (Thermo Fisher). Serum-starved and growth factor depleted conditions were utilized as a tool to stunt normal RMVEC tube formation stimulation to better decipher changes that would be observed through the addition of 100 nM Ang-(1–7). Treatment conditions included RMVECs plus vehicle and RMVECs plus 100 nM Ang-(1–7). At 24 and 48 hours 10X magnification images were taken using a TS100 Inverted Microscope (Nikon Corporation) for analysis of the mean tube length per field (μm) using open-access PipeLine tube formation analysis software[18 (link)]. The results were averaged across biological and technical replicates followed by 1-way ANOVA.