Endothelial Monolayer Formation from kMSCs

Kidney mesenchymal stromal cells (kMSCs, 2 × 10⁴ cells) were seeded in 1% gelatin coated 96 well plate. After 4 h when all the cells adhered to the plate, HUVECs (2 × 10⁴ cells) were added on top of the kMSCs, cultured in EGM2 (Lonza, Basel, Switzerland) medium. The cells were cultured for 2 days until a confluent endothelial monolayer formed. Next, cells were fixed with 4% PFA and 0.2% Triton-X100 in PBS for 10 min at room temperature, washed with PBS, and blocked for 1 h at room temperature in 3% normal goat serum and 2% BSA in PBS. Primary monoclonal Mouse Anti-Human VE cadherin (CD144, 55-7H1, BD Biosciences, San Diego, CA, USA) and phalloidin-TRITC (P1951, Sigma, Zwijndrecht, the Netherlands) were incubated overnight at 4 °C, followed by an appropriate secondary antibody and Hoechst 33528 for 1 h, all in blocking buffer. Cells were examined using a LEICA TCS SP8 X WLL (Leica, Rijswijk, The Netherlands) and a 60x objective (HC PL APO CS2 40x/1.30 OIL, Leica). Sequential 16-bit confocal images (xyz dimensions, 0.142 × 0.142 × 0.3 μm) were recorded using LAS-X Image software (Leica) and analyzed with ImageJ. The stable linear adherence junctions were quantified as ratio over total junction length [23 (link),24 (link)].