In situ cell death detection kit

To assess the effect of folate deprivation on DNA fragmentation, treated cells were stained using terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick end labeling (TUNEL) with an in situ cell-death detection kit (Promega_ Diagnostic Corporation, Madison, WI, USA). Briefly, according to the manufacturer’s guidelines, cells were fixed with paraformaldehyde for 20 min at 4 °C. Subsequently, cells were permeabilized with 0.2% Triton X-100 for 15 min at RT. After removing the permeabilization solution, cells were incubated with equilibration buffer (EB) for 5 min at RT. Then cells were resuspended in 50 μl of rTdT incubation buffer (45 μl EB + 1 μl rTdT enzyme + 5 μl nucleotide mix) and incubated in a water bath for 60 minutes at 37 °C in the dark. The reaction was stopped with 1 ml of 20 mM EDTA. After that, cells were washed with PBS⁻ containing 0.1% Triton X-100 ® and 5 mg/ml BSA. Finally, cells were analyzed with a FACS- Calibur™ flow cytometer (Becton Dickinson, San Jose, CA).

In Situ Cell Death Detection Kit (Promega Diagnostic Corporation, Mannheim, Germany), known as TUNEL (TdT-mediated dUTP-digoxigenin nick end labeling), was used for detection of apoptotic cells in blastocysts [10 (link)]. Hatched blastocysts were thoroughly washed in PBS + 1 mg/ml polyvinyl alcohol (PVA). Then, they were fixed in humid condition for 1 h at room temperature using 4.0% paraformaldehyde (w/v) in PBS. Following fixations, blastocysts were washed again in PBS/PVA and permeabilized for 30 min at room temperature, in 0.5% (v/v) Triton X-100 and sodium citrate. Next, permeable blastocysts were incubated for 10 min at room temperature in EQ buffer. Subsequently the blastocysts were incubated at 37 °C for 1 h in the dark under humid condition in TUNEL reaction mixture (equilibration buffer, nucleotide mix, and rTdT enzyme). Then, the blastocysts were allowed to remain in Buffer 2X for 15 min at room temperature. Eventually, the blastocysts were counterstained to label all nuclei with propidium iodide (PI) for 15 min, washed extensively in PBS, mounted on microscopic slides and observed under a fluorescence microscope (Olympus, Tokyo, Japan). Total numbers of nuclei were counted by PI. Cells were considered as TUNEL positive if their nuclei showed light green fluorescence against the background of PI (Fig. 2c).

Apoptotic cells were detected using a TUNEL assay kit (Promega Corporation, Madison, WI, USA). Fresh kidney sections were fixed in 10% buffered formalin and embedded in paraffin, and 4-µm slices were stained using the in situ Cell Death Detection kit (Promega Corporation) according to the manufacturer's instructions. Three tissue sections from each sample were randomly selected and 10 microscopic fields per section were assessed by two independent observers. In each field, the nuclei were quantified and the percentage of TUNEL-positive nuclei was determined.