Hello, Bo!

Various assays can be used to assess cytotoxicity on Сaсo-2 cells:

Trypan blue assay:

Article Hypoxic stress, hepatocytes and CACO-2 viability and suscept...

6. Viability assay: Hepatocytes and Caco-2 cells cultures in normoxic and hypoxic conditions were washed three times with PBS, and detached from wells using Trypsin-EDTA solution. The cells' suspensions were centrifuged at 1500 rpm for five minutes and the supernatants were removed. The cell pellets were resuspended in 1 mL PBS. After this the cells were stained with Trypan blue and counted in a Neubauer chamber.

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4.4. Cytotoxicity Assays

B. cereus cell-free supernatants were prepared from a 15 h liquid culture in Luria–Bertani broth medium (Oxoid, Dardilly, France) at 30 °C, under agitation (120 rpm). This culture was centrifuged (4 °C, 15 min, 9000× g) and then filtrated with a 0.22 µm porosity filter. Strain NVH1230-88, known to produce at least the Nhe, Hbl and CytK toxins, was used as positive control. The negative control consisted on the same strain (NVH1230-88) for which the toxins were degraded by heat treatment (80 °C, 20 min).

After 21 days in 48-well plates, Caco-2 cells (or Caco-2 and HT-29 cells) were washed with phosphate buffered saline (PBS) (137 mM NaCl; 2.68 mM KCl; 1.14 mM KH2PO4; 8 mM Na2HPO4.2H2O; pH 7.2) (Sigma-Aldrich, St. Louis, MO, USA), incubated during 3 h with the B. cereus supernatants, diluted two times in HBSS buffer (Lonza, Basel, Switzerland) and then washed with PBS before assays.

To assess the cytotoxic effect of these supernatants several cytotoxicity assays were performed including ATP, NR and LDH assays. The first assesses the cell viability by the cytoplasmic ATP dosage. This quantification is based on the measurement of the luminescent signal generated by the conversion of luciferin by luciferase and the cytoplasmic ATP. The second assay determines the cell viability by the spectrophotometric determination of NR (3-amino-2-methyl-phenazine hydrochloride) taken up by viable cells and stored in their lysosomes [61]. The third test evaluates the lactate dehydrogenase (LDH) released by cells in necrosis [62]. For each experiment, several wells of each plate were dedicated to the positive (incubation with 1% of Triton X-100) (Sigma-Aldrich, St. Louis, MO, USA) and negative (incubation with Hank’s Balanced Salt Solution (HBSS), 100%) controls.

4.4.1. Neutral Red Accumulation Assays

The treated cells (after washings) were incubated for 3 h with NR (750 µL at 0.33 mg/mL). Cells were then washed with PBS and NR was extracted using 300 µL of extracting solution (50% ethanol: 1% acetic acid) (Merck, Darmstadt, Germany). After 5–10 min of plate agitation the absorbance was measured by spectrophotometry at 540 nm [63]. The results were then expressed as percentages of Caco-2 cells viability over cells incubated with HBSS (toxicity negative control). The mean of the replicates and standard deviation (SD) were calculated for each B. cereus supernatant (Table A1 and Table A3). For the monoculture assays, with the aim to facilitate the comparison with the LDH assay, a toxicity value (from 0 to 3) was attributed to each supernatant as follows: 0 (non-cytotoxic), % of viability higher than 70%; 1, % of viability between 50% and 70%; 2, % of viability between 20% and 50%; 3, % of viability less than 20% (Table 1).

4.4.2. LDH Release Assays

After the 3 h of exposure of the intestinal cells to the B. cereus supernatant, the lactate dehydrogenase activity released in the culture medium was assessed using the Cytotoxicity Detection Kit (LDH) (Roche, Basel, Switzerland). The liquid culture was centrifuged to remove the cell debris. Then, 1 µL of cell-free liquid culture was mixed with 99 µL of PBS in a 96 wells plate. 250 µL of Catalyst solution (containing diaphorase and NAD+) was mixed with 11, 25 mL of Dye solution (containing iodonitrotetrazolium and sodium lactate) and 100 µL of this reagent were added to each well. The reduction by the LDH enzyme of NAD+ to NADH was measured by spectrophotometry at 500 nm. The results of this test were expressed in percentage of Caco-2 cell mortality over cells treated with Triton X-100 (positive control). The mean over replicates as well as their SD were calculated for each supernatant (Table A1 and Table A3). Similarly to NR and for the Caco-2 monoculture assays, a toxicity value (from 0 to 3) was attributed to each B. cereus supernatant as follows: 0 (non-cytotoxic), % of mortality less than 20%; 1, % of mortality between 20% and 50%; 2, % of mortality between 50 and 70%; 3, % of mortality higher than 70% (Table 1).

4.4.3. ATP Content Assays

The intracellular ATP content was monitored to assess the cytotoxicity of B. cereus supernatant for the co-culture assays instead of NR. Indeed, the mucin produced by the HT-29 cells could interfere with the NR uptake. The ATP content was measured using the luminescent CellTiter-Glo® assay kit (Promega, Fitchburg, WI, USA). After the B. cereus supernatant exposition, the eukaryotic cells were rinsed twice with PBS. Then, 150 μL of HBSS were added to each well during 15 min at room temperature. The CellTiter-Glo® buffer was mixed with the CellTiter-Glo® substrate and 150 μL of this reagent mix was added to the wells. The plate was agitated during 2 min to homogenize the solution and accelerate cell lysis. In order to measure the highest signal possible, 200 μL of the cell solutions were transferred into a 96 wells white polystyrene plate, specific for luminescence measurements (Nunc™, Thermo Fisher Scientific, Waltham, MA, USA). The plate was shaken for 30 s and the luminescence recorded with a fluorimeter-luminometer (Fluoroskan Ascent™ FL, Thermo Fisher Scientific, Waltham, MA, USA) every 2 min during 20 min (0.5 s of integration time). This kinetics allowed identifying the time lapse where the maximum of luminescence was observed. The results of this test were expressed in percentage of Caco-2 cell viability over cells incubated with HBSS (negative control).

MTT assay:

Investigating the Responses of Human Epithelial Cells to Predatory Bacteria

https://www.nature.com/articles/srep33485

Cytotoxicity Assays

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.

Hi Alexandr, thank you for your answer! As for the neutral red assay and the MTT assay, do you think bacteria could interfere with the assay? I will definitely have a look at the LDH assay protocol and will try some of the assays you proposed.

MTT ASSAY IS THE BEST ONE FOR VAIBILITY AND TOXICITY.

Many assays such as MTT, and WST-1 can be used to evaluate the cytotoxicity assay.