MTT formazan

The cells were plated in 24 well culture plates (25000 cells/500 µl/well for all the cell lines except Raw264.7, which was seeded with 40,000 cells/well density) and incubated in CO₂ incubator. Next day, treatment was given according to the experimental requirement. Further, at respective time points, 50 µl MTT solutions from the Stock (5 mg/ml) was added and cells were incubated in CO₂ incubator in the dark for 2 hrs. The medium was removed and Formazan crystals formed by the cells were dissolved using 500 µl of DMSO followed by transfer in 96 well plate. The absorbance was read at 570 nm using 630 nm as reference wavelength on a Multiwell plate reader (Biotech Instruments, USA). Reduced formazan quantification was done with Formazan standard. At each respective time points cell numbers were counted with a Neubauer-improved counting chamber (Paul Marienfeld GmbH & Co. KG, Germany) under 10X objective, and 10X eye piece magnification with compound light microscope (Olympus CH30, Japan). After MTT incubation Formazan images were captured under bright field (20X objective) with inverted microscope (Olympus, Japan). All the parameters were recorded under similar experimental conditions.

The cellular viability of SH‐SY5H cells and organotypic hippocampal slice cultures was assessed using a mitochondria‐dependent dye for live cells (tetrazolium dye; MTT) to formazan, as previously described 37. Cultures are pre‐treated with increasing concentrations of the test compound and incubated at 37°C with MTT (0.2 mg/ml) for 1 hr. The medium was removed and the cells lysed with dimethyl sulfoxide (100 μl). The extent of reduction in MTT to formazan was quantified by measurement of optical density at 550 nm with a microplate rider.

The in vitro cytotoxicity assay of the purified protease of S. typhimurium against HT29 human adenocarcinoma cell line (Merck, Darmstadt, Germany) was performed on a 96-well plate by incubation for 24 h. Per 1 × 10⁵ HT29 cells per milliliter, fifteen micrograms of the enzyme were used. By the end of incubation, the percent of cell death of HT29 was assessed by the standard MTT assay^{16 (link)}. For negative blanks, physiological saline was used instead of the active protease preparations, while for blanks, a medium without cells was used. The idea of this assay is that the remaining surviving cells can convert the yellow tetrazolium MTT reagent into the purple formazan complex with a characteristic absorption at A₅₄₀ nm, therefore indicating HT29 viability. The intensity of the purple color is in direct relation to the number of surviving cells after exposure to the UcB5.
Furthermore, an assay of cell-damaging activity against RBCs (hemolytic activity) due to the purified enzyme was done. This was achieved by vortexing identical volume sizes of 15 µg protease/mL and 4% (v/v) washed human RBCs suspended in 0.1 M borate buffer, pH 7.5. Incubation was done at 37 °C for 90 min, thereafter, the quantity of released hemoglobin was measured colorimetrically. For comparison, a complete hemolysis treatment was done by mixing RBCs suspension with 1% (v/v) triton X-100 solution.
Also, an in vivo screening of cell-damaging activity was done and the LD₅₀ value was calculated. For this, BALB/c mice weighing 22–25 g were acclimatized to the laboratory conditions for one week and retained at relatively fixed nutritional and physical conditions. They were then divided into six groups of six per cage. The first group was represented as the universal blank group. Mice in this group were intraperitoneally inoculated with an equal volume of a heat-denatured enzyme preparation at a concentration of 60 µg/body weight. While the other five groups were intraperitoneally injected with the active protease preparation at various concentrations (60, 30, 15, 8, and 4 µg protease/body weight) in a total volume of 1 ml solution. Animals were then observed at time intervals throughout 48 h for the LD₅₀ calculation according to the method of Karber. Livers of affected and blank mice were removed instantly after death and fixed in 5% (v/v) glutaraldehyde then 1% (w/v) OsO₄ solution. Before dissection of a blank mouse, it was anaesthsized by the inhalant gas sevoflurane. Ultrathin sections of 70 nm were sliced by RMC ultramicrotome and loaded on standard-grade TEM support grids made-up of copper for examination under JEOL 1010 TEM.

For cell proliferation, cells at a density of 1.5 × 10⁵/well were seeded in 12‐well plates. After treatment, cells were harvested and determined cell number using trypan blue (Solarbio, C0040) stain by cell counting plate. For MTT assay, cells were seeded in 96‐well plate with a density of 5 × 10³ per well in 200 uL of complete media. After treatment, the cultures were washed with PBS (phosphate buffered solution, Solarbio, P1022) for three times. Cytotoxicity was assessed using MTT (3‐[4,5‐dimethylthiazol‐2yl]‐2,5‐diphenyltetrazolium bromide, Solarbio, M8180). MTT solutions 20 uL (5 mg mL⁻¹ in PBS) along with 200 uL of fresh, complete media were added to each well, and plates were incubated for 4 h. After incubation, add 200 µL DMSO (Solarbio, D8371) into each well. Wrap plate in foil and shake on an orbital shaker for 5 min. Occasionally, pipetting of the liquid may be required to fully dissolve the MTT formazan. Absorbances were measured using microplate reader at 490 nm and results were expressed as % viability which was directly proportional to metabolic active cell number. Percentage (%) viability was calculated as: Cell Viability (100%) = OD in treatment well/OD in control(PBS) well × 100.
For CCK8 assay, the cell proliferation was measured using the Cell Counting Kit‐8 (NCM Biotech, C6005). The transfected cells were plated into 96‐well plates at a density of 5 × 10³/well/100 µL. and then 10 µL of CCK8 solution was added to each well, followed by incubation for 2 h. The cell viability was determined by measuring the absorbance at 450 nm.