An immortalized HaCaT keratinocyte cell line (ATCC, Manassas, VA, USA) was cultured in DMEM with 10% heat-inactivated FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin in a humidified atmosphere with 5% CO2 at 37°C.
Hacat keratinocyte cell line
Manufactured by American Type Culture Collection
Sourced in United States
The HaCaT keratinocyte cell line is a spontaneously immortalized human keratinocyte cell line derived from adult skin. It is a widely used in vitro model for studies related to keratinocyte biology and skin-related research.
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Lab products found in correlation
Heat inactivated fetal bovine serum, by Welgene (2 mentions)
Rpmi 1640 medium, by Welgene (2 mentions)
Cytation 5, by Agilent Technologies (2 mentions)
Autoscratch, by Agilent Technologies (2 mentions)
Scratch app, by Agilent Technologies (2 mentions)
Dmem glutamax, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Harvard Bioscience (1 mentions)
Pen strep, by Harvard Bioscience (1 mentions)
Amphotericin b, by GE Healthcare (1 mentions)
Coptisine, by Merck Group (1 mentions)
Streptomycin, by Welgene (1 mentions)
Penicillin, by Welgene (1 mentions)
75 cm2 plastic flasks, by Corning (1 mentions)
7 protocols using hacat keratinocyte cell line
1
Culturing HaCaT Keratinocyte Cell Line
2
Cytotoxicity Assays on Cell Lines
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Cytotoxicity assays were performed with the HaCat keratinocyte cell line (ATCC), the 3T3L1 fibroblast cell line (ATCC) and the hCMEC/D3 endothelial cell line (kindly donated by Dr. P. O. Couraud (INSERM, Paris, France)). Cells were cultured in DMEM Glutamax medium (Dulbecco’s Modified Eagle Medium DMEM GlutaMAXTM—Gibco, Massachusetts, USA), supplemented with 10% (v/v) fetal bovine serum (Biochrom, Berlin, Germany), 0.1% Amphotericin B (GE Healthcare, Little Chafont, United Kingdom) and 1% Pen–Strep (penicillin–streptomycin, 100 IU m−1 and 10 mg mL−1, respectively) (Biochrom, Berlin, Germany). Culture and incubation were performed in a 5% CO2 humidified atmosphere and at 37 °C.
3
Coptisine Modulates Keratinocyte Viability
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HaCaT keratinocyte cell line was obtained from the American Type Culture Collection (Manassas, MD, USA), and maintained in RPMI 1640 medium (WelGENE Inc., Daegu, Republic of Korea) containing 10% (v/v) heat-inactivated fetal bovine serum (WelGENE Inc.), streptomycin (100 μg/ ml), and penicillin (100 Units/ml) at 37°C in a humidified atmosphere containing 5% CO 2 and 95% air. coptisine (C 19 H 14 NO 4 + , purity ≥ 98%), which was purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA), was dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich Chemical Co.), and diluted with cell culture medium to adjust the final treatment concentrations, prior to use in the experiments. For the cell viability study, HaCaT cells were treated with different concentrations of coptisine for 24 h, or pretreated with coptisine for 1 h, before H 2 O 2 (0.5 mM, Sigma-Aldrich Chemical Co.) treatment for 24 h. The cells were also treated with 10 μM ZnPP (Sigma-Aldrich Chemical Co.), a well established HO-1 inhibitor, for 1 h in the presence or absence of H 2 O 2 .
4
Culturing HaCaT Keratinocytes in RPMI 1640
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HaCaT keratinocyte cell line was obtained from the American Type Culture Collection (Manassas, MD, USA). The cells were maintained at 37 °C in an incubator with a humidified atmosphere of 5% CO2, and cultured in RPMI 1640 medium (WelGENE Inc., Daegu, Korea) containing 10% (v/v) heat-inactivated fetal bovine serum (WelGENE Inc.), streptomycin (100 µg/mL), and penicillin (100 Units/mL). PG was purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA), dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich Chemical Co.), and diluted with cell culture medium to adjust the final treatment concentrations, prior to use in the experiments.
5
Culturing HaCaT Keratinocytes for Experiments
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The immortalised human HaCaT keratinocyte cell line was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). This cell line has two p53 spontaneously transformed point mutations. HaCaT cells were cultured in DMEM supplemented with 10% FBS, 5% penicillin–streptomycin, and 5% glutamine, maintained in a humidified incubator at 37 °C with 95% air and 5% CO2. Cells were cultured to around 80% confluence in 75 cm2 plastic flasks (Corning, New York, USA). Cells were plated into plastic bottom black wall 96 well plate (Perkin Elmer) and left to reach a confluence of 70–80% before being used for experiments.
6
Scratch Assay for Keratinocyte Migration
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Scratch assay was performed as previously described using the HaCaT keratinocyte cell line purchased from American Tissue Culture Collection (ATCC). Experiments were performed as previously described. Briefly, 100,000 cells were seeded in 24‐well plates and allowed to adhere to the culture plate overnight. Cells challenged with bacteria were stimulated overnight prior to scratch; cells challenged with chemicals were stimulated 4 h prior to scratch using the Autoscratch (BioTek). Cells were placed in the Cytation 5 (BioTek) at 37°C with 5% CO2; images and quantitation were performed by the Scratch App (BioTek).
7
Scratch Assay in HaCaT Keratinocytes
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