Keratinocyte sfm 1x

The gingival epithelial cells were isolated as described in the literature.^36,37 The gingival tissues were exposed to the enzyme solution containing Trypsin-EDTA (0.25%) (Gibco-BRL, Carlsbad, CA, USA) and 2 mg/mL Dispase II (Roche Diagnostics, Roche Applied Science, Germany) for 5 min to remove the epithelial part of the tissue. The separated epithelial tissue was minced and incubated for 20 min in the same enzyme cocktail previously mentioned. After incubation, fetal bovine serum (FBS) (Gibco, Rockville, MD, USA) was used to neutralize the enzymatic action. The mixture was centrifuged at 1800 rpm for 5 min. The count was taken and approximately 2 X 10⁵ cells were yielded from each sample. The pellet of cells obtained was resuspended and cultured in Keratinocyte SFM (1X) (Gibco, Gaithersburg, MD, USA) in 6-well plates. After 3 days, the cells started to attach to the surface of the culture plates. After that, the media was replaced with fresh medium every alternate day. On the 8th day of culture, the cells become confluent in the culture wells. The confluent cells were split in a 1:2 ratio and expanded further. Cells from passage 2–4 were used for the experiments.