Testing therapeutics in cell-based assays: Factors that influence the apparent potency of drugs
Cells and media are prepared in 100 μl/well cell plates and incubated overnight. Drugs in 50 μl/well are transferred from drug dilution plates to cell plates using a 96-well manual benchtop pipettor for 1 h of contact. In the biosafety level-4 (BSL4) laboratory, EBOV in 50 μl/ well is transferred to the cell/drug plates using a 96-well manual benchtop pipettor for a final volume of 200 μl/well. At specific assay endpoints, cells are fixed and transferred to the BSL-2. Immunostaining was performed with a EBOV-specific antibody against VP40 and a fluorescent or chemiluminescent secondary antibody using a plate washer/Dispenser. Fluorescence is quantified on a plate reader. The HCI system (Operetta) is used to detect EBOV-positive cells and count cells with a nuclei stain (Hoechst 33342). In parallel, cytotoxicity assays (CellTiter Glo) with mock infected cells are performed at BSL-2. Luminescence is read on the Infinite® M1000 Tecan plate reader. Data are analyzed using GraphPad Prism and/or Columbus software (Operetta).