Ms 295 p

Dissociated hiPSC-CMs treated with compounds were replated onto 20× FN-coated CellCarrier96 Ultra Microplates (Perkin Elmer) at 1 × 10⁴ cells/well and incubated for 4 days. Then the cells were fixed with 4% PFA and stained with cTNT (Thermo, MS-295-P, 1:500 dilution) and goat anti-rabbit IgG (H + L) highly cross-adsorbed secondary antibody, Alexa Fluor 546 (Invitrogen, A-11035, 1:500 dilution). Images were acquired using an OperaPhenix imager (Perkin Elmer, Waltham, MA). Roundness was measured using Harmony High-Content Imaging and Analysis Software (Perkin Elmer).

CaMiRi were rinsed with phosphate buffered saline (PBS) and fixed with 2% paraformaldehyde (PFA) overnight at 4 °C. They were then rinsed again with PBS and permeabilized in 0.1% Triton-X in blocking solution (2% BSA in PBS) for 5 min. Microtissues were then incubated in blocking solution for 30 min. Primary antibody was diluted in a 1% BSA in PBS solution and was added and left for 3 days at 4 °C. Mouse IgG1 anti-cardiac troponin (Thermofisher MS-295-P) was used at a dilution of 1:100, while Rabbit IgG anti-Vimentin (Abcam AB16700) was used at a dilution of 1:500. After three consecutive washes in PBS of 1 hour each, secondary antibodies were applied for 1 day at 4 °C. Alexa Fluor® 488 goat anti-mouse IgG1 and Alexa Fluor® 555 goat anti-rabbit (Life Technologies) antibodies were used at a dilution of 1:1000 for cardiac troponin and vimentin respectively. After the removal of the secondary antibodies, nuclear staining was performed using a DAPI solution (1 µg/mL in PBS) for 15 min. The microtissues were then washed in PBS (3 × 1 hour) before mounting on glass slides using fluoro-safe mounting media (Dako, S3023). Mounted samples were allowed to cure for 1 day and then imaged using a confocal microscope (Zeiss LSM700 Confocal Microscope).

Cells were fixed in 4% PFA for 10 min at room temperature and washed in 1× PBS three times. Cells were incubated in primary antibody buffer (containing primary antibody, 1% BSA, 0.1% saponin and 1× PBS) for 1 h at 37°C. After that, cells were washed in 1× PBS three times, then they were stained with secondary antibody buffer (containing secondary antibody, 1% BSA, 0.1% saponin and 1× PBS) for 1 h at 37°C. Finally, cells were washed three times in 1× PBS then stained with DAPI (1:1000) and mounted with Flouromount-G (SouthernBiotech) for imaging. Primary antibodies were: anti-NKX2.5 (Developmental Studies Hybridoma Bank, PCRP-NKX2-5-3B4, RRID: AB_2618896, 1:500) and anti-CTNT (Thermo Fisher Scientific, MS-295-P, RRID: AB_61806, 1:1000). Secondary antibodies were Alexa Fluor 488 polyclonal antibody (Invitrogen, A-11094, RRID: AB_221544, 1:1000) and goat anti-mouse IgG (H+L) cross-adsorbed secondary antibody, Cyanine3 (Invitrogen, A10521, RRID: AB_2534030, 1:1000) (Table S6).

Plasma membrane rupture, a major characteristic of necrosis that is not observed during apoptosis in vivo, was evaluated by Evans blue dye uptake assay as described previously (14 (link)). Briefly, mice received a single intraperitoneal injection of Evans blue (10 mg/ml, A16774, Alfa Aesar, 100 μg/g body weight) 16 h prior to I/R. At 24 h post I/R, mice were sacrificed and perfused retrogradely with 10 ml PBS. The heart was harvested and embedded in optimal cutting temperature (OCT) compound (Sakura), snap frozen in liquid nitrogen, and cut into 5-μm cryosections. Heart sections were stained with mouse anti-cardiac troponin T (cTnT, MS-295-P, Thermo Scientific, 1:100) to identify necrotic cardiomyocytes (Evans blue⁺/cTnT⁺). The percentage of necrotic cardiomyocytes was quantified with ImageJ.

Samples were fixed using 4% PFA with 0.25% TritonX-100. The following primary antibodies were used: mouse anti-cardiac Troponin T (Thermo MS-295-P, 1∶200); mouse anti-myosin heavy chain (clone MF20, eBioscience 53-6503-82, 1∶200); mouse anti-sarcomeric α-actinin (clone EA53, Sigma A7811, 1∶200); rabbit anti-Isl1 (Abcam ab20670, 1∶100), rabbit anti-smooth muscle myosin heavy chain (Abcam ab53219, 1∶250), goat anti-Nkx2.5 (Santa Cruz Biotechnology sc-8687, 1∶100), chicken anti-vimentin (Millipore AB5733, 1∶500), mouse anti-Myl7 (Abcam ab68086, 1∶100), rabbit anti-Myl2 (Proteintech 10906-1-AP, 1∶100), rabbit anti-Ki-67 (Novus NB110-89719, 1∶100), and mouse anti-V5 (Invitrogen 46-0707, 1∶200). For Oil Red O staining, samples were incubated with isopropanol for 5 minutes, followed by a solution consisting of 3 parts Oil Red O solution (0.3% Oil Red O in 99% Isopropanol, Sigma) and 2 parts DI water for 5 minutes. After washing with water samples were stained with hematoxylin (Sigma), washed with water, and visualized.