Atcc crl 1658

NIH/3T3 (American Type Culture Collection, Rockville, MD, USA, ATCC^® CRL-1658™) is a continuous fibroblast cell line derived from NIH/Swiss mouse embryo cultures by the same method as the original random-bred 3T3 (ATCC^® CCL-92™) and the inbred BALB/c 3T3 (ATCC^® CCL-163™) [64 (link)]. The NIH/3T3 cells are highly sensitive to sarcoma virus focus formation and leukemia virus propagation. In confluent cultures, NIH/3T3 proliferation is contact-inhibited.

The mouse NIH/3T3 cells (mouse embryonic fibroblast, ATCC CRL 1658) were obtained from the American Type Culture Collection were procured from Bioresource Collection and Research Centre and maintained under conditions described by the ATCC. Further, using Lipofectamine 2000 system (Invitrogen). These cells were genetically modified to stably express the enhanced GFP reporter gene (NIH3T3-G cells), which were further expanded and enriched to over 0.95% by fluorescence-activated cell sorting.

The biocompatibility of both plant extracts was assessed on NIH/3T3 fibroblasts cell line (ATCC CRL-1658; American Type Culture Collection, Rockville, MD, USA) using the Sulforhodamine B (SRB) assay. The NIH/3T3 cells were exposed for 48 h to different concentrations (15.5, 31, 62.5, and 125 µg/mL) of tested extracts added to the cell culture medium, or to the vehicle alone (DMSO) used as the control [45 (link)].

MTT (3-(4,5-dimethyl thiazol-2yl)-2,5-diphenyl tetrazolium bromide) assay was employed to study the cytotoxic activity against HeLa (human cervical carcinoma, provided by Prof. Dr. Anwar Ali Siddiqui from Aga Khan University, Karachi, Pakistan), and mouse fibroblast 3T3 (ATCC^® CRL-1658, purchased from American Type Culture Collection, ATCC, Virginia, USA) cell lines. The cells (1 × 10⁵ /well) were plated in 0.2 mL of DMEM high glucose medium/well in 96-well plates. The cells were treated for 24 hours with test compounds in the range of 25, 50, 75, 100, and 200 μM concentrations, respectively. For the MTT assay, the medium from the wells was removed carefully after 24 hours treatment. Each well was washed with 1X PBS for 2–3 times, and 200 μL of MTT (5 mg/mL) was added in to media containing wells (1:10). The plates were incubated for 4 hours at 37°C, in a 5% CO₂ incubator. After incubation, MTT was removed and 0.1 mL of DMSO was added to each well and mixed by keeping on a stirrer. The presence of viable cells was visualized by the development of purple color due to formation of the formazan crystals. The plates were observed under spectrophotometer (Spectramax) and absorbance was taken at 545 and 570 nm for cancer and normal cell lines, respectively. Measurements were performed and the concentration required for a 50% inhibition of viability (IC₅₀) was determined graphically.

We purchased mouse fibroblasts NIH/3T3 (ATCC® CRL-1658™) cell lines from American Type Culture Collection (ATCC) CCL-1658. We chose the NIH/3T3 cells because they are widely used as an in vitro model for a tissue model. Therefore, if the material tested is cytotoxic on this cell, its use in therapy is discouraged [43 (link)–45 (link)]. This cellular lineage was grown in plastic flasks (25 cm²) with RPMI 1640 medium, supplemented with 10% inactivated fetal bovine serum and 1% antibiotic/antimycotic solution [43 (link)]. The cultures were incubated at 37°C in an atmosphere containing 5% CO₂ [43 (link)]. Medium was changed every 48 h, and when the culture reached confluence, the subculture was treated with Trypsin-EDTA, until complete release of the cells. The released cells were transferred to a new plastic flask or 96-well plates [43 (link)–45 (link)].