A431 (DSMZ ACC 91), A549 (DSMZ ACC 107) and MRC-5 (ATCC CCL-171) cells were cultured in Dulbecco´s Eagle Medium (DMEM, Thermo Fisher), supplemented with 10% fetal bovine serum (FBS) superior (Merck Millipore) and 1x penicillin/streptomycin (Sigma Aldrich). Cells were cultured in T75 cell culture flasks at 37°C in a humidified atmosphere with 5% CO2 and passaged every 3-4 days after reaching 80% confluence. Daudi cells were maintained in RPMI 1640 supplemented with 20% FBS and 1x penicillin/streptomycin. Cells were subcultured every 2-3 days and incubated at 37°C and 5% CO2. Ready-to-use NHEK cells (PromoCell) were utilized for cellular binding studies.
Nhek cells
Manufactured by PromoCell
Sourced in Germany
NHEK cells are normal human epidermal keratinocytes, which are the predominant cell type in the epidermis, the outermost layer of the skin. These cells play a crucial role in maintaining the skin's barrier function and homeostasis.
Automatically generated - may contain errors
4 protocols using nhek cells
1
Cell Culture Protocols for Cell Lines
2
NHEK Keratinocyte Cytotoxicity Assay
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Normal human epidermal keratinocytes (NHEK) cells from juvenile foreskin from pooled donors were purchased from Promocell (Heidelberg, Germany) and cultured according to manufacturer’s recommendations in Keratinocyt Growth medium 2 (Promocell, Heidelberg, Germany). Cytotoxicity of probiotic strains was assessed using the 2,3-Bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT, Sigma-Aldrich) cell viability assay. NHEK cells were seeded at a density of 5000 cells/well in 96-well plates and cultured until confluent. Overnight cultures of probiotic strains or S. aureus were added to the wells with or without NHEK cells at 106 CFU/well and incubated for 2 h at 5% CO2 and 37°C. Triton X-100 (0.5%) was used as a positive control.
3
Keratinocyte Response to Bacterial Stimuli
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Commercially available primary normal human epidermal keratinocytes (NHEK) cells (Promocell) were cultured under serum-free conditions, supplemented with Keratinocyte Growth supplements (Promocell) as per the manufacturer’s instructions. Live B. oleronius and C. acnes were incubated with NHEK cells at 1 × 103 CFU (calculated from overnight plating) for 24 hours. After 24-hour stimulation, RNA was extracted from cells by phenol-chloroform extraction, and qPCR was performed for KLK5 (Hs01548153_m1).
4
Co-culture of NHEKs with Psoriatic DMSCs
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Total of 1×105 adult pooled NHEK cells (Promo Cell, Heidelberg, Germany) were cultured in keratinocyte serum-free medium (Promo Cell) with epidermal growth factor plus bovine pituitary extract (Gibco, Gran Island, NY) and 1% antibiotics, and seeded at 1×104 cells/ml in 25 cm2 tissue culture flasks at 37°C in 5% CO2. The medium was changed daily until the cells were confluent. NHEKs were detached using 0.25% trypsin and 0.04% EDTA. Total of 3×105 NHEKs (2×105 cells/ml) were seeded onto Transwell plates at passages 2-4 to co-culture with 1×105 DMSCs from 12 psoriasis and 12 control (500μl cell suspension at a concentration of 2×105 cells/ml), respectively. NHEKs were detached using 0.25% trypsin-0.04% EDTA after 72 h of co-culture. Afterward, NHEKs were harvested for further analyses. NHEKs cultured without any DMSCs for 72h alone served as “Untreated NHEKs”. The NHEKs co-cultured with psoriatic DMSCs and control DMSCs were labelled as “P-DMSC-Treated NHEKs” and “C-DMSC-Treated NHEKs”, respectively.
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