Cell proliferation kit 1 mtt

1 × 10⁴ second passage G-MSCs per well of each experimental group were cultivated in 24-well culture plates, and their cellular counts were determined every day for 14 days.
MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazoliumbromide) test was conducted at 24 and 72 hours (MTT Cell Proliferation Kit-I, Roche Diagnostics GmbH, Mannheim, Germany) [24 (link)] to test the cells metabolic activity. No phenol-red serum-free medium (RPMI 1640, PAN-Biotech, Aidenbach, Germany) and 0.5 mg/ml MTT-labelling reagent was added to the G-MSCs cultures and left for 4 hours, followed by 1 ml of the Solubilization solution (37°C, 5% CO₂, overnight). The spectrophotometrical absorbance was recorded at 570 nm wavelength (MultiskanGO Microplate Spectrophotometer, Thermo Fisher). Metabolic activity was calculated using standard curves. The assays were conducted in duplicate and averaged.
Second passage 1.63/cm² G-MSCs were cultivated in 10-cm-diameter dishes for the CFUs assay. On the 14^th day, cell cultures were fixed using 100% methanol (ice-cold, for 10 min) and stained with 0.1% crystal violet for 10 min. CFUs were evaluated by two independent examiners, using phase-contrast inverted microscopy. Aggregations of ≥50 cells were counted as a colony.

RPMI-1640, DMEM, FBS and antibiotics were purchased from Pan Biotech GmbH (Aidenbach, Germany). MTT Cell Proliferation Kit I, X-tremeGENE 9 DNA transfection reagent, protease and phosphatase inhibitor cocktails were purchased from Roche (F. Hoffman-La Roche Ltd., Basel, Switzerland). Primary NFAT5 (sc-13035, rabbit) and AR (sc-166919, mouse) antibodies were purchased from Santa Cruz Biotechnology Inc. (Dallas, Texas, USA) and used with 1:200 dilution in immunoblotting. SIRT6 antibody (SAB4200254, mouse) was purchased from Sigma (Darmstadt, Germany) and used with 1:1000 dilution in immunoblotting. Primary SIRT1 (#8469, mouse), PARP1 (#9542, rabbit), myc (#2272, rabbit), Lamin A/C (#2032, rabbit) and Beta-Actin (#4967, rabbit) antibodies were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA) and used with 1:1000 dilution in immunoblotting. Secondary HRP-conjugated anti-rabbit (#7074) and anti-mouse (#7076) antibodies were also purchased from Cell Signaling Technology Inc. (Beverly, MA, USA) and used with 1:5000 dilution in immunoblotting. NaCl, glucose, tris, glycine, and tween-20 were purchased from Molekula Ltd. (Newcastle Upon Tyne, UK). All other chemicals were obtained from Sigma (Darmstadt, Germany), unless otherwise stated.

DSA-induced growth inhibition in the AML cell lines was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) Cell Proliferation Kit I (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s instructions. Molm-14 cells were harvested from culture and seeded in a 96-well cell culture plate at a density of 5 × 10³ cells/well and treated with vehicle (DMSO) or increasing concentrations of DSA (0–1000 pM) for 72 h in a 5% CO₂ incubator at 37 °C. HL-60 cells were harvested from culture and seeded in a 96-well cell culture plate at a density of 5 × 10³ cells/well and treated with vehicle (DMSO) or increasing concentrations of DSA (0–1000 pM) for 72 h. Subsequently, the cells were labeled with the MTT labeling reagent for 4 h followed by the addition of a solubilization buffer for ~12–16 h (overnight). The plate was then read using the μQuant microplate spectrophotometer (BioTek^® Instruments, Inc., Winooski, VT, USA) and the absorbance was read at 570 nm wavelength using 650 nm as the reference wavelength.

Cellular toxicity was assessed with MTT Cell Proliferation Kit I, Roche Applied Science, Indianapolis, IN, USA according the manufacturer's protocol. Indicated amounts of Aβ_1‐42 and GMP‐1 were added directly to the culture media and incubated for 24 hours.

To measure cell viability, six TE-1 cells containing PARs expression-regulating plasmids and an equal number of vehicle control cells were equally plated in 96-well plates. Cell viability was determined using the standard MTT dye uptake method. MTT (5 mg/mL) was added and the formazan crystals that formed were dissolved in 10% SDS and 0.01 N HCl. The absorbance was measured at 570 nm with reference to 640 nm using a microplate reader (Infinite^® 200 Pro, Tecan, Switzerland) Cell growth was assayed using the MTT Cell Proliferation Kit I (Roche, Shanghai, China) following treatment with the inhibitor for 6, 12, 24, 48 and 72 h.