Journal: Mediators of Inflammation

Article Title: HaCaT Cells as a Reliable In Vitro Differentiation Model to Dissect the Inflammatory/Repair Response of Human Keratinocytes

doi: 10.1155/2017/7435621

Figure Lengend Snippet: Proliferation and differentiation of HaCaT cells in low (0.07 mM) and high (1.8 mM) Ca 2+ -containing medium. (a) Proliferation of HaCaT cells plated at the same density (1.0 × 10 4 cells/cm 2 ) assessed by the MTT assay at 2, 6, 9, and 14 days of incubation. Values represent mean ± SD of three independent experiments. (b) Western blot analysis of the expression of keratinocyte (KC) differentiation markers (K10, K14, and involucrin) in HaCaT cells grown in low (a) and high (c) Ca 2+ -containing medium for 6 (A6 and C6) or 14 (A14 and C14) days. The relative intensities of band signals quantified by digital scanning densitometry are reported in the histogram; β -actin was used to normalize the results to protein content. This blot is a representative of three independent experiments. (c) Immunofluorescence staining of HaCaT cells, grown in low (A) and high (C) Ca 2+ -containing medium for 6 (A6 and C6) and 14 (A14 and C14) days, and of primary human KC, grown in low Ca 2+ -containing medium, with anti-K10 antibodies (magnification: 60x).

Article Snippet: HaCaT and primary keratinocytes, cultured on 8-well slide chambers, were washed with PBS, fixed, and permeabilized in ice-cold methanol for 5 min. First, cells were incubated in PBS containing 0.1% bovine serum albumin (BSA) for 10 min, then were incubated with primary antibodies against K10 (rabbit monoclonal IgG anti-cytokeratin 10; 1 : 200; Genetex, Irvine, CA, USA) for 60 min in PBS containing 1% BSA.

Techniques: MTT Assay, Incubation, Western Blot, Expressing, Immunofluorescence, Staining