Mtt reagent

MRC5 cells were treated with compounds for 48 h and MTT assay was performed as reported previously^{21 (link)}. Cells were incubated with MTT reagent (1.2 mM, Life Technologies, Eugene, OR, USA) for 2 h. Acidic isopropanol was used to dissolve the formazan crystals and cell viability was measured by a spectrophotometer (Bio-Rad, Hercules, CA, USA). WST-1 viability assay was performed on Vero E6 cells treated for 48 h with compounds, following the manufacturer’s instructions (Roche Diagnostics GmbH, Mannheim, Germany). The cytotoxicity assays were performed in triplicates.

The impact of the MVs on the viability of the cultured HeLa cells was determined using the MTT assay according to the manufacturer’s suggested protocol. Briefly, 1 × 10⁴ cells were seeded into the wells of a 96-well plate and grown for 24 h. The cells were then exposed to the MVs from Y. pseudotuberculosis ATCC 29833 or YPIII by replacing the media within the wells with fresh media containing MVs. The cells were grown for an additional 24 h in the presence of the MVs. Afterwards, 5 μg/ml of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazoliumbromide (MTT reagent, Life Technologies, USA) was added to each well and the plates were incubated at for an additional 4 h at 37 °C in the dark. After thoroughly removing the media, 400 μl of DMSO was added to each well and the plate was incubated with shaking (150 rpm) at room temperature for 15 min to allow the color to develop. The OD was measured at 550 nm and used as a proxy for the HeLa cell viability.

The impact of the bacteria on the 24 hour viability of the cultured eukaryotic cells was also determined with the MTT assay. After the 24 hour exposure, approximately 5 μg/ml of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazoliumbromide (MTT reagent, Life Technologies, USA) was added to each well and the plates were incubated at 37 °C for 2–4 hours in the dark. Subsequently, the media was thoroughly removed, about 400 μl of DMSO was added to each well and the plate was incubated with shaking (150 rpm) at room temperature for 15 minutes to allow the color to develop. The OD was measured at 540 nm and used as a proxy for the viability of the cultured mammalian cells.

L929 cells were cultured overnight in 12-well plates and used at 50% of confluence. After pre-treatment with zVAD (20 μM) (Millipore) for 1 h, the cells were incubated with Necrostatin-1 (10 μM) (Santa Cruz), SP600125 (20 μM) (Absource), or DMSO as control. Nineteen hours later, the cells were incubated with 1 ng/ml of MTT reagent (Life Technologies) for 2 h. Once MTT crystals were developed and controlled under light microscopy, they were dissolved in DMSO and quantified by measuring absorbance at 540 nm.