Celltiter glo luminescent cell viability assay

Proliferating cells were seeded at approximately 20 × 103 cells per 50 μL per well into 96 well plates using a MultiFlo FX multimode dispenser (BioTek). On reaching ~40% cell density, the cells were then incubated with varying concentrations (195 nM to 200 μM final) of test samples using a Biomek FXP Automated Workstation (Beckman Coulter). 48 hours post drug incubation, cell viability was determined using Promega’s CellTiter-Glo Luminescent Cell Viability Assay according to the manufacturer’s protocol on a plate reader (EnVision from PerkinElmer) in Luminescence mode. In each experiment, each unique condition (i.e., different sample type and concentration) was tested in triplicate. The percentage of test sample induced toxicity was calculated with respect to DMSO treated cells, and graphed to give dose response curves. IC50 values for each treatment were calculated using GraphPad Prism 8.0 Software.

At day 11 of differentiation, podocytes were trypsinized, re–plated in collagen I–coated 384-well plates at a density of 4’000–8’000 cells per well and recovered for 48 hours before the experiment. Treatment with 2.5μM thapsigargin (EMD Millipore) in a total volume of 30 μl, and in the presence of the individual compounds (see key resources table for library details) was applied for 96 hours. Each small molecule was tested in duplicate. Cell viability was determined by measuring relative ATP levels in an EnVision microplate reader (PerkinElmer) using the CellTiter–Glo® Luminescent Cell viability assay (see key resources table) according to the manufacturer’s protocol. Positive and negative control cells were DMSO alone and thapsigargin in DMSO, respectively. Compounds were considered as hits (protective effect on thapsigargin–induced cell death) when both samples individually passed the 3σ threshold (hit: replicate1 ≥ thapsigargingargin±3σ ≤ replicate2).