2.2. MTT Cell Viability and BrdU Cell Proliferation Assay

Cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT), which is based on reduction of the yellow MTT to purple formazan by living cells [17]. In 96-well plates, 8 × 10⁴ cells were seeded in 100 μL of DMEM/F12 per well and were exposed to different concentrations of Imatinib according to the experimental protocol. After 48 h of treatment, the medium was changed to fresh medium containing 1 mg/mL of MTT. Two hours later, 100 μL of DMSO was added in each well and the absorbance at 570 and 630 nM was determined. The percentage of cell viability was calculated using a formula [percentage viability = (average OD of sample/average OD of control) × 100].

K562 cell proliferation was determined using the colorimetric bromodeoxyuridine (BrdU), which measures the incorporation of BrdU, a thymidine analogue, into the DNA of proliferating cells. The BrdU assay used in this study was an ELISA-based assay that was performed as recommended by the manufacturer (Merck-Millipore, USA). Imatimib treated K562 cells or ABL sgRNA virus infected K562 cells were incubated for 36 h at 37 °C, the media were supplemented with 10 μM BrdU and incubated for an additional 12 h. The cells were then stained with a peroxidase-labeled antibody against BrdU, followed by TMB Peroxidase Substrate addition for 30 min and acid stop solution exposure. The absorbance of the samples at 450 nm with a reference wavelength of 540 nm was measured using a microplate reader.