LD50 on A549 cell line
Median Lethal Dose (LD50)
Human lung epithelial cell line A549 (ATCC, Manassas, VA) was cultured at 37 °C in 5% CO2 in Dulbecco's modified Eagle medium (DMEM; VWR International), supplemented with 10% of fetal bovine serum (Gibco) and 2 mM glutamine (Invitrogen). Cells were incubated overnight in a 96-well plate (VWR International) at 5e4 cells/well. Serial dilutions of AT mutants or WT-AT was then added to cells for 2 h at 37 °C, plates were centrifuged at 2000 rpm for 2 min, and 50 μL supernatant was transferred to a new 96-well plate. Cell lysis was measured as release of lactate dehydrogenase (LDH), using the CytoTox 96 nonradioactive assay kit (Promega) by following the manufacturer's recommendations. As a positive control, cells were lysed with 10% SDS. Background of LDH release was subtracted, and 100% of cell lysis was calculated as 100 × [(OD490 of cells with AT)/(OD490 of cells with SDS)].