Use of a colorimetric assay to measure differences in cytotoxicity of Mycobacterium tuberculosis strains

Abstract

Several techniques have been used to quantify the cytotoxicity produced by Mycobacterium tuberculosis bacilli on cell monolayers; however, they are semi-quantitative or time consuming. Herein, a method based on crystal violet (CV) uptake by THP-1 cell monolayers is described. This colorimetric method quantifies the cytotoxic effect as a function of the number of remaining cells after the infection with M. tuberculosis. Since this micro-organism is not stained by the dye, it does not produce a background that affects absorbance readings. As determined by CV assay (CVA), M. tuberculosis strain H37Rv destroyed 10.5 % of THP-1 cell monolayers at 24 h and 50.52 % at 72 h, while M. tuberculosis strains lacking the complete phospholipase C locus produced a reduced cytotoxic effect. The damage estimated by microscopy corresponded to the effect quantified by CVA. The results show that the use of CVA is a rapid, sensitive and reliable quantitative assay to measure the cytotoxicity of different M. tuberculosis strains. INTRODUCTIONMycobacterium tuberculosis is a bacterial pathogen that produces a detrimental effect on mammalian cell cultures. This effect can be due to the induction of cell apoptosis or necrosis leading to cell death. The phenotype is reported as a cytotoxic or cytopathic effect (Castro-Garza et al., 2002;Danelishvili et al., 2003;Dobos et al., 2000;McDonough & Kress, 1995) and it can be roughly quantified by analysing the altered cell morphology (cell rounding and loss of monolayer integrity) (Daniel et al., 2004;Fischer et al., 1996), setting up a scale to estimate the percentage of rounded or detached cells (Read et al., 1974) or counting the amount of degraded cells by electron microscopy (McDonough & Kress, 1995). A more precise quantitative assay, such as measuring lactate dehydrogenase (LDH) release by using a colorimetric kit, has also been reported (Danelishvili et al., 2003;Dobos et al., 2000). However, most of the above experimental procedures are only semiquantitative or are time-consuming.There are other methods to quantify cytotoxicity; however, they are not practical for use with M. tuberculosis infection systems: exclusion or inclusion of vital dyes requires direct handling of samples, the release of radiolabelled substances increases the biosafety level, and the reduction of coloured compounds such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), 4-[3-4(iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) and other tetrazolium salts by bacteria (Franzblau et al., 1998;Gomez-Flores et al., 1995) as well as macrophages (Ferrari et al., 1990) in in vitro infection models would produce a high background and the results obtained would not be accurate.Crystal violet (CV) is a triphenylmethane dye also known as gentian violet. The most commonly used application for this dye is as the primary stain in the Gram-staining procedure. Gillies et al. (1986) A unique characteristic of M. tuberculosis bacilli is their outer lipid bilayer, which is the t...

EsxA membrane-permeabilizing activity plays a key role in mycobacterial cytosolic translocation and virulence: effects of single-residue mutations at glutamine 5

“…7b). To measure the Mtb -induced cytotoxicity, we used the crystal violet uptake assay, which is a colorimetric method that quantifies the cytotoxic effect as a function of the remaining live cells after infection44. The reason why we quantified live cells instead of dead cells as described in Live/Dead assay (Fig.…”

Immune Modulation as an Effective Adjunct Post-exposure Therapeutic for B. pseudomallei

“…The plate was then read on a plate reader at 570nm and 600nm. This procedure was adapted and optimized from Kursheed et al [14] and Castro-Garza et al [15]. Each treatment had a technical replicate of 10 and the experiment was run in biological triplicate for statistical significance.…”

Intracellular activity of tedizolid phosphate and ACH-702 versus Mycobacterium tuberculosis infected macrophages

“…THP-1 cells were transformed to macrophages as described previously [18] and then incubated at concentrations 16X the MIC for M. tuberculosis H37Rv (ACH-702: 2 μg/ml, Tedizolid: 8 μg/ml, Rifampicin: 15 μg/ml and Moxifloxacin: 8 μg/ml). After 12 h exposure (time used in posterior experiments) viability was assessed using the crystal violet technique [18,19]. In all cases cell viability after drug treatment was greater than 98%.…”