Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich) assay or trypan blue exclusion assay. MTT assay was performed as previously described56 (link). Briefly, cells (2.5 × 103 cells/well) were seeded in each well of a 96-well plate and incubated for 24 h. The cells were cultured in amino acid-depletion media for an additional 72 h. Thereafter, 50 μL of MTT (2 mg/mL in PBS) was added to each well and the plates were incubated for an additional 2 h. The resulting formazan crystals were dissolved in 100 μL of dimethyl sulfoxide (DMSO) after aspiration of the culture medium. Plates were placed on a plate shaker for 1 min and read immediately at 570 nm using a TECAN micro-plate reader with Magellan software (Tecan Group Ltd., Männedorf, Switzerland).
The trypan blue exclusion assay was performed by seeding 1 × 105 cells/well into 12-well cell culture plates and incubating them for 24 h at 37 °C. Seventy-two hours after P4HA3 siRNA transfection, cells were disaggregated in 500 μL medium and 10 μL of the suspension was mixed with 10 μL trypan blue (Thermo Fisher Scientific). Viable cell counts were obtained using the Countess Automated Cell Counter (Thermo Fisher Scientific).
The trypan blue exclusion assay was performed by seeding 1 × 105 cells/well into 12-well cell culture plates and incubating them for 24 h at 37 °C. Seventy-two hours after P4HA3 siRNA transfection, cells were disaggregated in 500 μL medium and 10 μL of the suspension was mixed with 10 μL trypan blue (Thermo Fisher Scientific). Viable cell counts were obtained using the Countess Automated Cell Counter (Thermo Fisher Scientific).
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