Er id red assay kit

After respective treatment cells were seeded on 8 well Lab-Tek Chambered coverglass (Thermo, Rochester, NY) and fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X100, stained with Hoechst 33342 reagent. The ER-ID Red Assay Kit (Enzo Life Sciences, Lörrach, Germany) was used for staining of the ER according to the manufacturer's instructions. Samples were mounted and photographed under an immunofluorescence microscopy. Quantify the fluorescence density of NCTD treated cells with ER-ID Red Assay Kit by FACSCalibur flow cytometer and the data were analyzed by Cell Quest software (BD Bioscience, Bedford, MA).

Endoplasmic reticulum (ER)-specific fluorescence intensity, indicating ER expansion, a hallmark of activated ER stress, was measured using the ER-ID Red assay kit (Enzo Life Science, Farmingdale, NY, USA). Cells were seeded in a 60 mm or 96-well plate culture dish. After a 24 h incubation, DWP05195 was diluted with the culture medium and added to the cells. Following another 24 h incubation, 100 µL 1X Assay Buffer with 1 µL of ER-ID Red Detection Reagent and 1 µL of Hoechst 33342 Nuclear Stain was added into each wells or dishes, and the cells were incubated for 30 min at 37 °C. The fluorescent intensity was analyzed by flow cytometry and cells were imaged using a confocal fluorescence microscope.

Staining of SR in HiPSC-CMs exposed to H/R was performed using ER-ID® Red assay kit (GFP-CERTIFIED®) (Enzo Life Sciences, cat # ENZ-51026-K500). The experiment was performed as per the manufacturer’s instructions. In brief, live cells in 4 well chamber slides were stained using 1× staining solution containing 1 µl of ER-ID Red Detection Reagent in 1 ml of 1× assay buffer provided in the assay kit for 30 min at 37 °C protected from light. Cells were washed twice with PBS, followed by fixation with 3.7% PFA, and mounted with DAPI mounting solution. Images were captured using a Leica SP8 confocal microscope at 63× magnification objective. The fluorescence signal for ER-ID® Red was detected at ex. 561 nm and emission at 590 nm. Laser intensity for the 561 nm line was maintained at 7%. DAPI line 405 nm was used at 40% for clear nuclei identification and counting purposes. Image acquisition settings were kept constant for the complete experiment. Fluorescence intensity quantification for ER-ID® Red and nuclei count was performed using NIH ImageJ software.