Preparation of primary human keratinocyte.

Primary human keratinocytes were obtained from fresh tissue of excised neonatal foreskin after in­formed consensus. After removal of the subcutane­ous tissue and much of the reticular dermis, the tissue samples were cut into 2 x 2 mm strip and incubated in 0.05% Tripsin, 0.53 mM ETDA solu­tion overnight at 4 C. The following day, the epidermis was peeled off the residual dermis and aspirated using a Pasteur pipette to aid cell disso­ciation. The primary suspension of primary epider­mal cells was washed with PBS (Phosphate Buffered Saline, pH7.4) twice then prepared in Epilife basal culture medium (Cascade Biolibics Inc., Portland, Or, USA) with Human keratinocyte growth supple­ment (Cascade Biolibics Inc., Portland, Or, USA).

Subculture and treatment with TNF-a and IFN-y

Prepared primary keratinocytes were seeded into culture plates and maintained at 37 C in a humidified incubator with 5% CO2 until forming the monolayer then trypsinized and cells were plated in secondary culture (5 x 104 cells/cm2) and serially subcultured at every 60-70% confluence. Cells from each passage were seeded at 6-well culture plates (3 x 105 cells/well) and cultured in presence or absence of 10 ng/mL of TNF-a (R&D Systems Inc, Minneapokis, USA) and/or IFN-y (R&D Systems Inc, Minneapokis, USA) for another 24 hours.  Apcalis Oral Jelly

HaCaT cell culture and treatment with TNF-a and IFN-y

The human keratinocyte cell line HaCaT was cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and 100 y/mL penicillin/streptomycin at 37°C and in 5% CO2. Forming monolayer cells were trypsinized, then seeded at 6-well plates (1 x 105 cells/well). At approximately 70% of confluence, 10 ng/mL of TNF-a and/or IFN-y were treated and cells were cultured for another 24 hours.

Growth pattern analysis

Proliferation patterns of cultured keratinocyte was calculated using an inverted phase-contrast micro­scope and cells were collected at planned time for counting the number with hemocytometor. The number of population doublings was calculated according to the previous report. generic cialis 10 mg

Where Nt was the number of viable cells at the end of the growth period and N was the number of cells attached to the flasks after plating.