Cell viability assays for metabolic inhibitor experiments

RBCs were isolated from donors as mentioned above. The RBC pellet was made up to 9 ml with Krebs-Henseleit Buffer. 420 µl of RBCs were aliquoted into the wells of a 96 deep well plate for drug addition. 2.1 µl of diluted drug (1:200 dilution; 0.5% DMSO final) was added to each well and mixed thoroughly. For 6AN, concentrations were screened from 0 to 10 mM. For heptelidic acid, concentrations were screened over the range of 0 to 1 mM. Fifty microliter of RBCs preparation with compounds added (and control RBCs with only 0.5% DMSO added) were aliquoted into 96-well plates. The plates were incubated at 37 °C in constant darkness. Sampling was then performed every 24 h over a period of 96 h. At each time point, RBCs in a plate were re-suspended by pipetting up and down. The 96-well plates were then spun for 5 min at 375 × g and 25 µl of the supernatant transferred into a 384-well plate for assaying. A standard curve was prepared by lysing untreated cells with water (hypotonic lysis) and then performing serial dilutions in a 96-well plate. The absorbance of the sample supernatants was measured at 480 nm (in a 384-well plate) and using a Tecan M1000 plate reader. Red cell absorbance measurements were acquired using Tecan M1000 plate reader software.