Nih 3t3 fibroblast cells

Human colorectal carcinoma cells, HCT116, HT29 and SW480, were obtained from the American Type Culture Collection (ATCC, Manassas, VA). HCT116 and HT29 cells were cultured in McCoy's 5A, and SW480 cells were cultured in RPMI-1640 supplemented with 10% fetal bovine serum (Hyclone, Logan, UT). NIH3T3 fibroblast cells were also purchased from the ATCC and cultured in DMEM supplemented with 10% calf serum (Invitrogen, Carlsbad, CA).

Cytotoxicity of test compounds on NIH-3T3 fibroblast cells (ATCC, Manassas, VA, USA) was evaluated by using the standard MTT colorimetric assay. Briefly 100 μL of 5 × 10⁴ cells/mL in Dulbacco’s modified eagles medium (DMEM) supplemented with 10% foetal bovine serum (FBS) were plated into 96-wells flat bottom plate and incubated overnight at 37 °C in 5% CO₂. Three different concentrations of test compound (1, 10 and 100 µg/mL) were added to the plate in triplicates and incubated for 48 h. 0.5 mg/mL MTT (50 µL) was added to each well, the plate was then further incubated for 4 h. MTT was aspirated and 100 µL of dimethyl sulfoxide (DMSO) was then added to each well. The extent of MTT reduction to formazan within cells was calculated by measuring the absorbance at 540 nm, using spectrophotometer (Spectra Max plus, Molecular Devices, CA, USA). The cytotoxic activity was recorded as concentration causing 50% growth inhibition (IC₅₀) for 3T3 cells. Cycloheximide was used as standard drug.

NIH/3T3 fibroblast cells were purchased from ATCC, USA, and maintained in a Petri dish with DMEM/F12 containing 10% fetal bovine serum (GIBCO, USA) and 1% penicillin-streptomycin (Solarbio, China). When the cell confluence area reached 80% in the Petri dish, cells were detached from the dish using trypsin (Solarbio, China). Cells were cultured under conditions of 37°C and 5% CO₂ until the confluence area of cells was up to 80% in the Petri dish. Then, trypsin was used to separate the 3T3 cells from the substrate of the dish. After centrifugation and discarding the supernatant, the cell precipitate was diluted in deionized (DI) water supplemented with 5% glucose and 2% bovine serum albumin (Solarbio, China) before manipulation.