Normal human epidermal keratinocytes purchased from Kurabo Industries (Osaka, Japan) were cultured in serum-free keratinocyte growth medium, HuMedia-KG2 (Kurabo Industries) containing human epidermal growth factor (0.1 ng/mL), insulin (10 mg/mL), hydrocortisone (0.5 mg/mL), gentamycin (50 mg/mL), amphotericin B (50 ng/mL), and bovine brain pituitary extract (0.4% vol/vol). After the cells had been serially passaged at 60–70% confluence, experiments were conducted using subconfluent cells at passage 3 or 4 in the proliferative phase at 60–80% confluence. After medium removal, the cells were washed twice with phosphate-buffered saline (PBS) before culture for 24 hours in HuMedia supplemented only with antibiotics. Keratinocytes were subsequently stimulated with YKS.
Normal human epidermal keratinocytes
Manufactured by Kurabo
Sourced in Japan
Normal human epidermal keratinocytes are primary cells derived from human epidermis. They serve as a model system for studying keratinocyte biology and function.
Automatically generated - may contain errors
Lab products found in correlation
Humedia kg2, by Kurabo (1 mentions)
Bovine pituitary extract, by Kurabo (1 mentions)
Insulin, by Kurabo (1 mentions)
Gentamicin, by Kurabo (1 mentions)
Collagen, by Merck Group (1 mentions)
Amphotericin b, by Kurabo (1 mentions)
Epilifetm medium, by Thermo Fisher Scientific (1 mentions)
Glass bottom dishes, by Iwaki (1 mentions)
Hydrocortisone, by Kurabo (1 mentions)
Humedia kg, by Kurabo (1 mentions)
Epilife medium, by Thermo Fisher Scientific (1 mentions)
12 well plate, by BD (1 mentions)
Il 13, by BioLegend (1 mentions)
Recombinant human il 4, by R&D Systems (1 mentions)
Humedia kb2, by Kurabo (1 mentions)
Il 17a, by R&D Systems (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Il 22, by R&D Systems (1 mentions)
Ifn γ, by R&D Systems (1 mentions)
4 protocols using normal human epidermal keratinocytes
1
Serum-free Culture of Epidermal Keratinocytes
2
Newborn vs. Adult Keratinocyte Comparison
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Normal human epidermal keratinocytes were purchased from Kurabo (Osaka, Japan). To compare the response of keratinocytes from newborn babies and adults, four different batches of newborn keratinocytes vials from different donors (0 years–1, 2, 3 and 4) and two different batches of adult keratinocytes vials (donors aged 40 years and 57 years) were used (details are summarized in Supplementary Table 1 ). Cells were cultured in EPILIFETM medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with insulin (10 μg mL−1), human recombinant epidermal growth factor (0.1 ng mL−1), hydrocortisone (0.67 μg mL−1), gentamicin (50 μg mL−1), amphotericin B (50 ng mL−1), and bovine pituitary extract (0.4%, v/v), all of which were sourced from Kurabo, at 37 °C in a CO2 incubator. Undifferentiated keratinocytes between passage 2 and passage 6 were used for experiments. EPILIFETM medium contains Mg2+, but the concentration is not disclosed.
For fluorescence imaging, keratinocytes were seeded on glass bottom dishes (IWAKI, Shizuoka, Japan) coated with 5 μg mL−1 collagen (Sigma-Aldrich, Saint Louis, MO, USA) at a concentration of 6–8 × 104 cells mL−1.
For fluorescence imaging, keratinocytes were seeded on glass bottom dishes (IWAKI, Shizuoka, Japan) coated with 5 μg mL−1 collagen (Sigma-Aldrich, Saint Louis, MO, USA) at a concentration of 6–8 × 104 cells mL−1.
3
Paprika Oil Effects on Keratinocytes
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Normal human epidermal keratinocytes were obtained from Kurabo (Osaka, Japan) . Keratinocytes were cultured at 37℃ in EpiLife™ medium (MEPI500CA, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with growth factors (Humedia-KG, Kurabo) . Commercially available red paprika xanthophyll oil was used (PapriX-oil) for the treatment of keratinocytes. The paprika oil was diluted to each concentration with dimethyl sulfoxide. At sub-confluence, cultured keratinocytes were stimulated with 0.05％, 0.005％, and 0.0005％ paprika oil. After 24 h, cells were collected and used for qRT-PCR and Western blot analyses.
4
Cytokine-induced gene expression in NHEK
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Normal human epidermal keratinocytes (NHEK) were purchased from Kurabo Industries (Osaka, Japan). NHEK were cultured in 75 cm 2 cell culture flasks (Sigma-Aldrich, St Louis, MO, USA) at 37°C, 5% CO 2 in Humedia-KB2 (Kurabo Industries) supplemented with Human Keratinocyte Growth Supplement sets (Kurabo Industries). The cells received fresh medium every 3 days and were subcultured every 10 days. When 80% confluence was achieved, the cells were trypsinized, washed, and resuspended in the medium at 5×10 5 cells/ml, and 1 ml was added to each well of the 12-well plates (Becton Dickinson Labware, Franklin Lakes, NJ, USA). When the cells reached confluence, the medium was completely removed and 1 ml serum-free medium was added to each well. Simultaneously, recombinant human IL-4 (R&D Systems, Minneapolis, MN, USA) IL-13 (BioLegend, San Diego, CA, USA), IL-17A (R&D Systems), IL-22 (R&D Systems) and IFN-γ (R&D Systems) were added, and the cells were incubated at 37°C and 5% CO 2 . The concentration of IL-17A was 1, 10, or 100 ng/ml, IL-4 and IL-22 were 10 or 100 ng/ml, IL-13 was 0.2, 2, or 20 ng/ml, and IFN-γ was 0.1, 1 or 10 ng/ml. After 24 h, cells were processed by TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) for isolation of total mRNA, according to the manufacturer's instructions.
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