Assessing Cell Viability with Calcein AM

Cell viability assays were performed using our previously described method^{24 (link)}. Briefly, an EZViable Calcein AM Fluorometric Cell Viability Assay Kit (BioVision, Milpitas, CA, USA) was used to quantify the number of viable cells. MRMECs were cultured on sterile black 96 well plates under growth conditions. At 75% confluence, MRMECs were treated with 0–0.5 µM AS-Eng shRNA–lipid in complete medium, 0 to 0.5 µM NS-shRNA–lipid in complete medium or 70% ethanol as a positive control for 8 h. After treatment, the cells were washed with cold PBS (Life Technologies Corporations). MRMECs were then exposed to a buffered (1:500) calcein AM solution and incubated at 37 °C for 30 min. Fluorometric readings were performed using a microplate reader (Biotek; Winooski, VT). Fluorescence intensity was plotted on the Y-axis and represented as % live cells. Experiments were performed at least three times with n = 3 for each experimental group.