Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL) assay

In Situ detection of apoptotic cells were performed using a DeadEnd^TM Fluorometric TUNEL System (Promega, Madison, WT). The assay was performed in accordance with the manufacturer’s protocol. Briefly, rMC-1 cells were grown on chamber slides and treatments were given as mentioned above. The cells were then fixed with 4% paraformaldehyde for 15 minutes at 4°C. After permeabilization with 0.1% Triton X-100 for 2 minutes, cells were incubated with the reaction mix containing terminal deoxynucleotidyl transferase (TdT) and nucleotides for 1 hour at 37°C. 4’,6-diamidino-2-phenlindole (DAPI) was used to stain the nuclei. Five nonoverlapping fields in each well were captured using a light microscope (Eclipse 80i Nikon, Tokyo, Japan) equipped with a digital camera (Diagnostic Instruments, Inc., Sterling Heights, MI). More than 250 cells in each field were counted using Image J software (National Institute of Mental Health, Bethesda, MD). Cells with TUNEL labeling co-localized with DAPI staining were counted as TUNEL-positive. The results were expressed as percentage of TUNEL-positive cells over the total number of cells counted from five individual experiments with duplicate samples.