Cell death detection assay

Primary ECs were seeded on coverslips in a 24-well-plate precoated with gelatin. After 24 hrs., cells were treated with PBS or exosomes isolated from NRVFs treated with either BSA or TGF-β1. After 48 h. of the treatment, cells were washed with PBS and fixed with 4% paraformaldehyde (PFA). Apoptotic cells were determined by TUNEL staining as per manufacturer's instructions (Cell death detection assay; Roche, Indianapolis, IN). ECs were counter stained with CD31 antibodies before TUNEL staining. DAPI was used to stain the nuclei.

TUNEL staining was carried out on 4-μm thick paraffin-embedded sections by using cell death detection assay (Roche). TUNEL⁺ nuclei showed green color. Cardiac myocytes were identified using sarcomeric α-actinin antibodies (MilliporeSigma). DAPI staining was used to count the total number of nuclei and to colocalize TUNEL-stained nuclei. The index of apoptosis was calculated as the percentage of apoptotic myocyte nuclei to total number of nuclei.
Alternative measure of apoptosis was carried out to detect the activated caspase-3 in formalin-fixed paraffin-embedded heart sections by SignalStain apoptosis (cleaved caspase-3) IHC detection kit (catalog 12692, Cell Signaling Technology). Images were obtained using an Olympus whole slide scanning microscope with an original total magnification of 200×.

For apoptosis analysis in transfected RCC cells a cell death detection assay (Roche) was performed. 5 × 10⁴ cells were seeded in a 6-well-plate. Next day medium was discarded; cells were scraped off and transferred in 500 μl incubation buffer. After 30 min at RT a centrifugation at 20000 g for 10 min followed. The supernatant was used for cell death detection assay. For the assay the 96-well-plate was incubated with 100 μl coating solution per well over night at 4 °C. Next day the solution was discarded, 200 μl incubation buffer was added per well and incubated for 30 min at RT. A washing step with 300 μl washing solution per well followed for three times. 100 μl of the prepared supernatant was then filled in the wells. After 90 min the solutions were discarded and wells were washed again (3 × 300 μl/well washing solution). Incubation with 100 μl per well coating solution for 90 min at RT followed. After another washing step apoptosis was detected with 100 μl substrate solution per well. The adsorption of individual wells was measured at 405 nm with GloMax®-Multi detection system (Promega). Experiments were performed in quadruplicates and repeated three times. Mean value and standard error rate were calculated.