Cytotox 96 non radioactive cytotoxicity kit

The specific killing activity of anti-HIV CAR-T cells toward Jurkat cells, LE6 cells, LEL6 cells, ACH-2 cells or HIV-1 infected primary CD4⁺ T lymphocytes at different ratios from 10:1 to 0.5:1 was measured by lactate dehydrogenase assay (LDH assay) using the CytoTox 96 non-radioactive cytotoxicity kit (Promega, Madison, WI, United States). The manufacturer’s instructions were followed. Briefly, 1 × 10⁴ target cells were co-cultured with anti-HIV CAR-T cells at various E:T ratios in a 96-well U-bottom plate and incubated for 8-24 h at 37°C. The supernatants were collected and incubated with 50 μL Cytotox 96 regent for 30 min. 50 μL Stop solution were added to each well followed by quantification of absorbance at 490 nm.
The cytotoxic effects of anti-HIV CAR-T cells on LEL6 cells were also confirmed by luminescence detection. CAR-T cells were co-culture with LEL6 cells (1 × 10⁴ cells) at different ratios from 10:1 to 1:1, 8 h later, 100 μL of Glo reagent (Promega) was added to each well and incubated for 10 min. Then, luciferase activity was measured by an inspired plate reader (Biotek, Montpelier, VT, United States), reflecting the cytotoxicity effects.

Microglia were seeded in 96-well plates at a concentration of 7 × 10⁴ cells/well and incubated overnight. Microglia were then treated with BzATP (300 μM), ATP (5 mM), or nigericin (20 μM) for 30 min at 37 °C. Untreated microglia served as the negative control while lysed cells served as the positive control. Fifty microliters of cell supernatant was collected and used to detect LDH activity with the CytoTox96 Non-Radioactive Cytotoxicity Kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions.

Caspase-3 activity was determined as an indicator of apoptosis. Detached cells were washed from plates with 500 μl PBS and collected by centrifugation. Adherent cells were lysed using a buffer containing 5 mM Tris HCl pH 7.4, 5 mM EDTA and 5% NP-40 and the lysate removed to the tube containing the corresponding cell pellet. Samples were frozen at –20°C until needed for processing. A 50 μl aliquot of the cell lysis was added to 1 ml of caspase substrate buffer containing 50 mM HEPES, 10% sucrose and 0.1% CHAPS with 10 mM DTT and 1 μl/ml of the caspase-3 fluorescent substrate Ac-DEVD-AFC (Calbiochem, EMD Millipore). Reactions were incubated at room temperature overnight prior to measurement of caspase activation using a spectrofluorimeter with excitation at 400 nm and emission at 505 nm. Apoptosis was also measured by Annexin-V and propidium iodide staining followed by flow cytometry using an Annexin-V FLUOS kit (Roche). Lactate dehydrogenase (LDH) release, as a measure of cytotoxicity, was determined using the Cytotox 96^® Non-Radioactive Cytotoxicity Kit (Promega, Madison, WI).

The cytotoxicity of the selected compounds was determined using mouse macrophages as prototypes of host cells. RAW 264.7 macrophages (10⁵ cells) were cultivated in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and 0 to 10 μM of the selected compounds (100-μL suspensions distributed into the wells of 96-well plates). The plates were incubated for 24 h at 37°C with 5% CO₂. The supernatant was recovered and tested for lactate dehydrogenase activity using the Cytotox 96 Non-Radioactive Cytotoxicity kit (Promega) according to the manufacturer’s recommendations. Control systems included vehicle (1% DMSO)-treated cells (viability control) and macrophage extracts using the lysis solution provided by the manufacturer (death control). From this point on, we selected compound MMV1593537 for subsequent tests, due to its low toxicity to the cultured macrophages.