Ldh release assay kit

An LDH Release assay kit (Cat #04744926001, Roche) was used to determine cellular viability after exposure to gentamicin. Cells were plated in a 96-well, flat bottom plate, grown to confluency, and then treated with gentamicin for the respective amounts of time used in internalization and binding assays. A positive control was generated by the treatment of cells with 5 µL of the provided lysis buffer for 15 min at 37°C. About 50 µL of cell supernatant was then removed from cells and added to a separate 96-well, flat-bottom plate and 50 µL of the catalyst and dye mixture (1:45 dilution of catalyst to dye) was added to all wells. The plate was incubated for 10 min at 37°C and the reaction was stopped by adding 25 µL of stop solution to each well. Absorbance was measured at 490 nm and 620 nm (to measure background) on a SpectraMaxi3x plate reader (Molecular Devices). 620 nm values were subtracted from 490 nm values to remove background signal and then a media only (no cells) condition was subtracted from all conditions. Viability was calculated by taking the ratio of sample to lysed control and multiplying by 100 to obtain % viability.

Cytotoxicity (cell wall integrity) was determined by a flow cytometry using propidium iodide (PI) staining or by using LDH release assay kit (Roche Applied Science). After 24 h of incubation with extracts/compounds, the neutrophils or HUVECs were harvested and centrifuged (1,500 RPM; 10 min; 4°C), washed once with cold PBS and re-suspended in 400 μL of PBS. Five microliters of PI (50 μg/mL) solution was added to the cell suspensions. After 15 min of incubation at room temperature, cells were analyzed by cytometry, and 10,000 events were recorded per sample. Cells that displayed high permeability to PI were expressed as a percentage of PI(+) cells. After 24 h of incubation with extracts/compounds, with monocyte/ macrophage cells, the supernatants were harvested and the LDH level was measure according to manufacturer's instruction. The percentage of death cells was calculated based on the effect of Triton X-100 (100% of cytotoxicity).