Tcs sp5 aobs laser scanning confocal microscope

Roche In Situ Cell Death Detection Kit (Sigma 11684795910) was used to visualize the viability of cells in frozen sections of cardiac organoids based on the Roche protocol (TUNEL Staining). TCS SP5 AOBS laser scanning confocal microscope (Leica Microsystems) was used for imaging. Quantification of viability was achieved using ImageJ particle analysis. Briefly, we quantified the overall number of nuclei represented per organoid through DAPI staining, followed by a quantification of the total number of TUNEL-positive nuclei for the corresponding organoid. We then used the complementary percentage of the ratio between TUNEL-positive nuclei to total nuclei to estimate the percentage of viable cells per organoid cross-section, defining the “Viability Index” for each organoid culture condition. To study the effect of culture conditions on cell viability, the changes of viability (Δ% Viability Index) in each culture condition compared to the ischemic condition were calculated for each organoid size group. A similar method of quantification was used for the staining and analysis of phospho-histone H3 (PHH3, Millipore 06–570). PHH3 was co-stained with DAPI, and the percentage of PHH3-positive nuclei per organoid cross-section was estimated using ImageJ. For each culturing condition and time-point during the 1–3 day time point, we quantified at least n=5 organoids.

Roche In Situ Cell Death Detection Kit (Sigma) was used to visualize the viability of cells in frozen sections of cardiac organoids based on the Roche protocol. Briefly, cardiac organoid frozen sections were fixed with 4% paraformaldehyde in PBS for 20 min at room temperature. Following washing in PBS for 30 minutes, samples were incubated in a permeabilization solution (0.1% Triton X-100 and 0.1% sodium citrate in PBS) for 2 minutes on ice. Then 50 μl of the TUNEL reaction mixture were added to samples and incubated at 37 °C for 1 hr. After washing in PBS (2 times at 5 min), nuclei were counterstained with DAPI (Molecular Probes/Invitrogen) diluted in PBS for 15 min at ambient temperature. Following the final wash procedure (PBS, 2 times at 5 min), glass cover slips were added to the slides using Fluoro-Gel (Electron Microscopy Sciences). TCS SP5 AOBS laser scanning confocal microscope (Leica Microsystems) was used for imaging. TUNEL-based viability index was calculated as [1 – (TUNEL-positive area/DAPI-positive area)].