A human immortal keratinocyte line (HaCaT), human skin fibroblasts (HSFs) and human embryonic kidney 293T cells (HEK 293T) were obtained from KeyGEN Biotech (Nanjing, China). Cells were cultured in DMEM (Gibco, USA) supplemented with appropriate fetal bovine serum (FBS) and 100 U/mL penicillin G and grown in 5% moist CO2 at 37 °C. HaCaT and HEK 293T cells were supplemented with 10% FBS, while HSF cells were supplemented with 15%.
Hacat
Manufactured by Keygen Biotech
Sourced in China
HaCaT is a cell line derived from human skin keratinocytes. It is a widely used in vitro model for studying various aspects of keratinocyte biology, including cell proliferation, differentiation, and response to external stimuli.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Caski, by Keygen Biotech (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Hek293t, by Keygen Biotech (1 mentions)
Rpmi 1640, by Keygen Biotech (1 mentions)
Dmem medium, by Keygen Biotech (1 mentions)
Caski, by American Type Culture Collection (1 mentions)
Hacat, by American Type Culture Collection (1 mentions)
Hyclone, by GE Healthcare (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
Dual luciferase assay system, by Promega (1 mentions)
Qpcr detection kit, by Merck Group (1 mentions)
Non targeting control sirna, by GenePharma (1 mentions)
8 protocols using hacat
1
Culturing Human Cell Lines for Research
2
Comparative Cell Culture Techniques
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The human normal cervical cell lines (Hacat) and cervical cancer cell lines (HeLa, Siha, Caski, and C33a) were purchased from American Type Culture Collection (ATCC, USA). HeLa, Siha, C33a, and Hacat cells were incubated in DMEM medium (KeyGEN, Nanjing, China), and Caski cells were cultured in RPMI1640 (KeyGEN, Nanjing, China) medium containing 10% fetal bovine serum (GIBCO-BRL, Invitrogen, Carlsbad, CA, USA) and cultured at 37°C in a humidified incubator containing 5% CO2.
3
HaCaT Cell Culture Protocol
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HaCaT, the human keratinocyte cell line, purchased from Jiangsu KeyGEN BioTECH Corp., Ltd. (Jiangsu, China), which was grown in the Central Laboratory of Shanghai Sixth People's Hospital Affiliated to Shanghai Jiaotong University (Shanghai, China), and cultured in Dulbecco's modified Eagle's medium (HyClone; GE Healthcare Life Sciences, Logan, UT, USA) supplemented with 10% heat inactivated fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA). All cells were maintained in a humidified incubator at 37°C with 5% CO2.
4
HaCaT Cell Line Cultivation Protocol
5
Psoriasis-like in vitro model
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The cell line HaCat purchased from KeyGEN Biotech (Jiangsu, China) was maintained in RPMI1640 with 10% FBS. The cell was cultured with 5% CO2 at 37 °C. When HaCat cells grew to 30–40%, cytokines (50 ng/ml TNF-α, 20 ng/ml IFN-γ, 30 ng/ml IL-17, and 30 ng/ml IL-22) were added to the culture solution to simulate the inflammatory environment of psoriasis in vitro. GSK126 dissolved in DMSO was used at the concentration of 50 nM. The HaCat cell line was authenticated by STR profiling and tested for mycoplasma contamination.
6
Cervical Cancer Cell Line Characterization and Luciferase Assay
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Human cervical cancer cell lines SiHa, CaSki, Hela, Me180 and human non-tumor keratinocyte line HaCaT were obtained from Nanjing KeyGen Biotech Co, Ltd (Nanjing,China). The cells were cultured in Dulbecco’s modified Eagle’s medium (GIBCO, Carlsbad, California, USA) containing 10% FBS in a humidified atmosphere of 5% CO2 at 37 °C. Human TFF3 expression, TFF3 siRNA and CDH1 siRNA plasmid constructs have been previously described [7 (link), 17 (link)]. Luciferase assays were performed as previously described [18 (link)]. Briefly, transfections were carried out in triplicate using 1 μg of the appropriate luciferase reporter construct and empty vector per transfection along with 0.1 μg of Renilla luciferase construct as control for transfection efficiency. Luciferase activities were assayed after 24 h of transfection using the Dual Luciferase Assay System (Promega Corp, Madison, WI, USA).
7
Authentication and Maintenance of Melanogenic and Keratinocyte Cell Lines
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A human melanogenic cell line (A375) and a human immortal keratinocyte line (HaCaT) were obtained from KeyGen Biotech (Nanjing, China) in 2013 and authenticated by short tandem repeat genotyping. Mycoplasma was analyzed using a Mycoplasma quantitative polymerase chain reaction (qPCR) detection kit (Sigma) before experiment. Cells were grown in DMEM supplemented with 10% FBS and maintained at 37 °C in a humidified atmosphere containing 5% CO2.
8
Keratinocyte Knockdown and Overexpression
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This study was approved by the Ethics Committee of Xijing Hospital. Written informed consent was obtained from all donors. Human keratinocyte cell line HaCaT purchased from KeyGEN Biotech (GB300, Nanjing, China) was cultured in RPMI 1640 supplemented with 10% fetal bovine serum at a humidified atmosphere at 37 C with 5% CO 2 .
siRNA and lentiviral RNA For transient knockdown of Trim21, three siRNAs for human Trim21 gene were used (Trim21 siRNA). Nontargeting control siRNA was used as a negative control (Genepharma, Shanghai, China). siRNAs for K17 were designed and synthesized by Ribobio Biotechnologies (Guangzhou, China). siRNAs were transfected into cells by using the Lipofectamine 3000 transfection reagent according to the manufacturer's instruction. siRNA sequences for K17 and Trim21 are listed in Supplementary Table S2 online.
For stable overexpression of Trim21 and K17, lentiviral Trim21 (LV-Trim21), lentiviral K17 (LV-K17), and lentiviral control RNA (LVcontrol) were constructed (Genepharma). Lentivirus production was carried out following the manufacturer's instructions. Cells were selected with 10 mg/ml puromycin. The lentiviral particles were titered by immunostaining of Trim21 in HaCaT cells infected with limiting dilutions of virus. The minimum number of viral particles required for 100% infection of keratinocytes was calibrated for experiments.
siRNA and lentiviral RNA For transient knockdown of Trim21, three siRNAs for human Trim21 gene were used (Trim21 siRNA). Nontargeting control siRNA was used as a negative control (Genepharma, Shanghai, China). siRNAs for K17 were designed and synthesized by Ribobio Biotechnologies (Guangzhou, China). siRNAs were transfected into cells by using the Lipofectamine 3000 transfection reagent according to the manufacturer's instruction. siRNA sequences for K17 and Trim21 are listed in Supplementary Table S2 online.
For stable overexpression of Trim21 and K17, lentiviral Trim21 (LV-Trim21), lentiviral K17 (LV-K17), and lentiviral control RNA (LVcontrol) were constructed (Genepharma). Lentivirus production was carried out following the manufacturer's instructions. Cells were selected with 10 mg/ml puromycin. The lentiviral particles were titered by immunostaining of Trim21 in HaCaT cells infected with limiting dilutions of virus. The minimum number of viral particles required for 100% infection of keratinocytes was calibrated for experiments.
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