In situ direct dna fragmentation tunel assay kit

To assess apoptosis following n-3 PUFA and/or PAL treatment(s), TUNEL assay was performed using an In Situ Direct DNA Fragmentation (TUNEL) Assay Kit (Abcam, Cambridge, United Kingdom, ab66108) according to the manufacturer’s instructions. Briefly, cell smears after paraformaldehyde fixation, blocking and permeabilization were incubated with TUNEL reaction mixture for 1 h at 37°C protected from light and counterstained with RNase/PI solution for 30 min. Cells were imaged using the EVOS FL Auto 2 Cell Imaging System at 20× magnification. A total of 10 fields were randomly selected for imaging, and for each treatment condition TUNEL positive cells relative to total cells in these fields were counted (Life Technologies, Carlsbad, CA, United States). For all treatment conditions, parameters such as brightness and contrast were kept consistent during the acquisition and analysis of all images. Analysis was performed by converting images to 16-bit grayscale images and applying the threshold function within ImageJ software.

TUNEL assay was performed with an In Situ Direct DNA Fragmentation (TUNEL) Assay Kit (ab66108) from (Abcam) according to the manufacturer’s instructions as described previously (19 (link)). Briefly, the cells were seeded in dishes and incubated overnight in a humidified chamber to adhere. Cells were or were not treated with Salubrinal (25 µM), Rapamycin (100 nM), and Chloroquine (50 µM) for 2 h. Cell monolayers were then washed thrice with DPBS and exposed to UV-B (20 and 30 mJ/cm²). Again, the cells were washed thrice with DPBS and supplemented with fresh DMEM media with or without the indicated concentrations of Salubrinal, Rapamycin, and Chloroquine as required and incubated further for 6 h post-UV-B irradiation. Cell smears after fixation, blocking, and permeabilization were incubated with TUNEL reaction mixture for 1 h and wrapped in aluminum foil to avoid light exposure at 37°C and counterstained with RNase/PI solution for an additional 20 min. Substrate solution was added, and cells were imaged by a fluorescent microscope (EVOS FL Colour Imaging System) for the detection of TUNEL-positive cells, and the intensity of fluorescence was quantified using the ImageJ software.