Calcein am pi cell viability cytotoxicity assay kit

H9C2 cells were cultured in 96-well plates. Given different treatments, they were cultured in assay buffer mixed with 1 µM calcein acetoxymethyl ester and 1 µM PI per well at 37°C for 30 min according to the introduction of Calcein-AM/PI Cell Viability/Cytotoxicity Assay Kit (Beyotime Biotechnology Inc., China). Green fluorescence by Calcein-AM indicates living cells. Red fluorescence by PI represents dead cells. The images were acquired with a fluorescence microscope (Leica Microsystems, Germany). The percentage of positive cells was counted with Image J software and each group had more than 10,000 cells.

Mouse tissue samples were taken, washed, and cut into 1 mm³ tissue pieces, which were then digested with 0.1% trypsin, filtered with a 40 μm pore size filter, and centrifuged to collect the precipitated cells. The cells were resuspended in a medium without phenol red and serum. Calcein-AM/PI Cell Viability/Cytotoxicity Assay Kit (C2015M, Beyotime) was applied for measuring the cell viability rate. Cells were mixed with 1× Assay Buffer, and dyed by 2 μM Calcein-AM and 4.5 μM PI at 37 °C for 30 min. A fluorescence microscope (Olympus IX51) was utilized for observing and Image Pro advanced software for evaluating the average fluorescence intensity [29 (link)].
The tissues were prepared into 4-μm-thick sections before experimentation. The apoptosis in cells and tissues was detected using TUNEL Fluorometric Kit (Promega, Madison, WI), with DAPI (D8200, Solarbio, Beijing, China) utilized for nuclear staining. Images were acquired with a fluorescence microscope at a magnification of ×400, and positive cells were counted at a magnification of ×200, with at least ten fields of view checked for each sample [29 (link)].