Cell Culture and Cytotoxicity Test Using Alamar Blue and MTT Assay

The THP-1 cell line was offered by ATTC for this study. VACSERA, a holding business for biological products and vaccines in Cairo, Egypt, provided the HepG2 and the MCF-7 cell lines. THP-1 cells were grown in RPMI 1640 medium, which included 10% heat-inactivated fetal bovine serum, 1% glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin. On a 96-well plate, cells were seeded at a density of 10,000 cells/well before being treated with different amounts of purified B. pseudomallei L-asparaginase and incubated for 48 h at 37°C in 5% CO₂. Untreated cells were seeded in the same circumstances as the treated cells, in a 20 mM potassium phosphate buffer (pH 7.5). Following incubation, each well received 10 µl of alamarBlue reagent (10% alamarBlue, Invitrogen, USA), and incubation was maintained at 37°C for another 4 h. The absorbance of the plates was measured at 570 nm for the plates and 600 nm for the reference using a microplate reader. The percentage of cell viability was expressed relative to the control cells after blank normalization [25]. Morphological changes in THP-1 cells were explored and documented using phase-contrast optical microscopy at a magnification of 40. The HepG2 and MCF-7 cell lines were cultured in DMEM high glucose media (4.5 g/l) supplemented with 10% FCS and 1%penicillin/streptomycin at 37°C and 5% CO₂. For the 3-(4, 5-Dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide reagent (MTT) test, cells were seeded at a density of 10,000 cells/well in a 96-well plate. The media were replaced after 24 h with a new mixture containing different concentrations of B. pseudomallei L-asparaginase, which was cultivated for 48 h. The cells were incubated for 3 h at 37°C in 5% CO₂ after being given MTT (5 mg/ml in 1 PBS). The cells were centrifuged and incubated in 100 µl of DMSO after incubation. After agitating the plates for 5 min, the absorbance was measured at 490 nm. For both treated and untreated cells, the proportion of viable cells was measured as control and plotted against B. pseudomallei L-asparaginase concentrations to calculate the IC₅₀ [26].