Cytotox 96 nonradioactive assay kit

LDH assay was performed using Promega Cytotox 96 Non-radioactive assay kit according to the manufacturer’s protocol. Cytotoxicity is calculated using the following formula Experimental-Target Spontaneous-Effector Spontaneous/Target Maximum-Target Spontaneous LDH Release × 100%.

To determine the cytotoxicity of the compounds, 70 to 80% confluent 293T cells in a 96-well plate were incubated with serial dilutions of each compound (up to 100 μM) for 5 h. Cell cytotoxicity was determined by a CytoTox 96 nonradioactive assay kit (Promega, Madison, WI) by measuring a cytosolic enzyme lactate dehydrogenase, which is released upon cell lysis, following the manufacturer’s instructions, and the 50% cytotoxic concentration (CC₅₀) value for each compound was calculated using GraphPad Prism software. The nonspecific cytotoxic effects of these compounds were also reported previously (30 (link), 31 (link), 40 (link), 43 (link), 44 (link)).

Human cervical carcinoma cells (HeLa cells) were routinely cultured and passaged in T-75 flasks with Dulbecco's Modified Eagle Medium (DMEM; Gibco Life Technologies)/10% Fetal Bovine Serum (FBS; Gibco Life Technologies)/1% antibiotics (penicillin/streptomycin) and incubated at 37°C with 5% CO₂. Cells were grown to 70–80% confluency and passaged approximately every 3–4 days and carried to a maximum of 25 passages from original stocks. Cells were washed with calcium-magnesium-free phosphate-buffered saline (CMF-PBS) prior to treatment with trypsin and before assays. For in vitro cytotoxicity assays, HeLa cells grown in a multi-well round-bottom plate format were washed with sterile CMF-PBS, inoculated with bacteria for an MOI (multiplicity of infection) of 50, or mock inoculated with medium alone and incubated for 4, 8, or 24 h. After the appropriate incubation period, cytotoxicity was measured via lactate dehydrogenase (LDH) release using the Cyto Tox 96 Non-Radioactive Assay kit (Promega). All work was carried out in a sterile class II laminar flow hood.

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 2,000 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)].

Neurotoxicity was induced by exposure of the neuronal cell cultures to mono-sodium glutamate (Na-glutamate). Briefly, hippocampal neurons, 9 days in culture, were exposed to 10 min of 5 μM filter-sterilized Na-glutamate. Plates exposed to glutamate were co-incubated with test compounds and placed in a humidified incubator at 37°C and 5% CO₂. To terminate glutamate exposure, cells were washed three times in modified Neurobasal media (composition: 1% B27, 1% Pen/Strep, 0.5% glutamine). Thereafter, compounds were added back to each well so that the final concentration of the drug was the same during the glutamate exposure and overnight incubation. Twenty-four hours after exposure to Na-glutamate, cell death was quantified by measuring the release of lactate dehydrogenase (LDH) in the culture media using a CytoTox-96 non-radioactive assay kit (Promega, Madison, WI, United States). Neuroprotection was calculated as the percentage reduction in cell death induced by exposure to Na-glutamate for 10 min.

LDH release was measured using the CytoTox 96® Non-Radioactive assay kit (Promega). Briefly, cells were plated in the 96-well U bottom plate in phenol red-free medium and treated with cell death inducing agents or media (control). Post treatment, the plates were spun at 250 g for 4 minutes. Supernatants were carefully transferred into a flat bottom plate followed by the calorimetric detection of LDH activity as described in the manufacturer protocol.