Fluorometric tunel kit

Cells were plated at a density of 50,000 per well in 24-well plate and cultured overnight. The following day, the cells were deprived of FBS overnight to standardize the cell cycle followed by fresh DMEM containing 10% FBS and the indicated concentration of glucose. At the specified times, cells were harvested using 0.5% trypsin (Invitrogen) and washed with wash buffer (0.1% FBS in PBS) twice to remove trypsin. Cells were fixed in ice-cold 70% ethanol for 45 min at 4 °C. Ethanol was removed by washing twice with PBS, and the cells were incubated with propidium iodide (PI) (40 μg/ml) and RNase (10 μg/ml) for 30 min at 37 °C. Samples were analyzed using Beckman Coulter FC500 Flow Cytometry Analyzer. Cell apoptosis was analyzed using flow cytometry (BD LSR II, San Jose, CA, USA) with annexin-V and propidium iodide staining and Fluorometric TUNEL kit purchased from Promega.

Cells containing fragmented DNA were evaluated in paraffin sections using a fluorometric TUNEL kit (Promega, Madison, WI, USA, G3250) according to the manufacturer's protocol. Briefly, the sections were dewaxed in xylene and rehydrated through a decreasing series of ethanol concentrations in water. The sections were immersed in 0.85% NaCl for 5 min, washed in PBS, fixed in 4% paraformaldehyde in PBS for 15 min and washed twice in PBS. To permeabilize the sections and allow antigen unmasking, the slides were treated with hot (>85 °C) citrate buffer for 10 min and then cooled for 20 min before being washed with PBS. The sections were incubated with 100 μl of equilibration buffer for 10 min and then labeled with 50 μl of TUNEL reaction mix for 1 h at 37 °C. The reactions were stopped by a 15-min wash in × 2 saline-sodium citrate buffer. The sections were washed in PBS to remove excess label and then dyed with 5 μg ml⁻¹ Hoechst 33342 (BD Biosciences) for 30 min at room temperature. After washing in PBS, the slides were mounted with Vectashield, and the images were collected using a CoolSNAP ES Olympus BX51 camera with the associated Metaview Software (Tokyo, Japan).

Cell viability in tissue sections was detected using a TUNEL fluorometric kit (Promega, NSW, Australia) according to the protocol supplied by the manufacturer. The slides were deparaffinised in xylene then rehydrated by immersion through a series of graded alcohols, washed in 0.85% v/v NaCl then PBS, fixed in 4% v/v paraformaldehyde in PBS, washed in PBS, and permeabilised in 20 μg/mL proteinase K (22 °C; 10 min).
Next, the slides were washed in PBS then fixed in 4% v/v paraformaldehyde, washed in PBS, and incubated with equilibration buffer (22 °C; 10 min) then with rTdT incubation buffer [90% Equilibration Buffer, 10% Nucleotide Mix, 2% rTdT Enzyme (37 °C; 1 h)]. Reaction was stopped by immersion in 2xSSC, slides were washed in PBS, and nuclei were counterstained in 1:600 Spectral DAPI (PerkinElmer, USA) in TBST. Fluorescent slides were imaged on the ZEISS AXIO SCOPE upright fluorescent microscope (Zeiss, NSW, Australia) and the level of cardiomyocyte viability was quantified using MetaMorph^® image analysis software (version 7.6; Molecular Devices, San Jose, CA, USA). An intensity threshold was manually selected to include all fluorescent apoptotic cells. The intensity of fluorescence was measured as integrated OD and normalised against the section area.

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)].