Human keratinocytes (HaCaT) were commercially procured from Shanghai EK-Bioscience Biotechnology Co., Ltd. HaCaT cells and the isolated splenocytes from mice were maintained in Dulbecco's modified eagle's medium (DMEM, Sigma Aldrich) containing 10% fetal bovine serum (FBS; F2442, Sigma Aldrich). A humidified incubator with 5% CO2 at 37°C was used to incubate the cells. The isolated splenocytes were resuspended in DMEM (Sigma Aldrich) supplemented with 10% FBS (Sigma Aldrich). Splenocytes were stimulated with anti-CD3 (1 lg/ml; BD pharMingen, San Diego, CA, USA) or anti-CD3 plus interleukin (IL)-6 (20 ng/ml; peproTech, London, UK) and transforming growth factor-β (TGF-β; 1 ng/ml; PeproTech) in the presence or absence of triptolide for 4 d.
Hacat
Manufactured by Merck Group
Sourced in United States, Germany
HaCaT is a well-established immortalized human keratinocyte cell line that is widely used in dermatological research. It is a robust and stable cell line that retains many of the characteristics of normal human keratinocytes.
Automatically generated - may contain errors
Lab products found in correlation
Dulbecco modified eagle medium (dmem), by Merck Group (7 mentions)
Hacat, by American Type Culture Collection (6 mentions)
Fetal bovine serum (fbs), by Merck Group (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Roswell park memorial institute (rpmi), by Merck Group (2 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (2 mentions)
Transforming growth factor β tgf β, by Thermo Fisher Scientific (1 mentions)
F2442, by Merck Group (1 mentions)
Anti cd3, by BD (1 mentions)
Saos 2, by Merck Group (1 mentions)
Mccoy s 5a medium, by Merck Group (1 mentions)
Rpmi medium, by Merck Group (1 mentions)
Penicillin, by Merck Group (1 mentions)
Streptomycin, by Merck Group (1 mentions)
Hff 1, by American Type Culture Collection (1 mentions)
Hacat, by Cosmo Bio (1 mentions)
F12 medium, by Merck Group (1 mentions)
Hacat, by BNCC (1 mentions)
16 protocols using hacat
1
Regulation of T cell activation by triptolide
2
Osteosarcoma and Keratinocyte Cell Lines
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The human osteosarcoma cell lines U2OS (p53 wt) and SAOS-2 (p53 null) and a human keratinocyte cell line (HaCaT) were purchased from the Rio de Janeiro Cell Bank (BCRJ, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil). U2OS and SAOS-2 cells were grown in McCoy’s 5A medium (Sigma-Aldrich®, St. Louis, MO, USA), while HaCaT cells were grown in RPMI medium (Sigma-Aldrich®), supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 µg/mL streptomycin (Sigma-Aldrich®). The cells were cultured at 37 °C in a humidified atmosphere of 5% CO2 for all experiments. Chalcones D14 and D15 (Figure 1 A) were provided by Dr. Luis Octávio Regasini (Department of Chemistry and Environmental Chemistry, São Paulo State University, São Paulo, Brazil).
3
Culturing Human Skin Cell Lines
4
Cell Culture Protocol for Cervical Dysplasia and Cancer
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The human cervical dysplasia cell line W12 (RRID:CVCL_T290, clone 20863),22 which was authenticated using short tandem repeat (STR) profiling, was gifted by Drs Paul Lambert, Tomomi Nakahara, and Iwao Kukimoto and used in this study. This cell line contains HPV16 episomes. W12 cells were cultured at 37°C under 5% CO2 in F medium composed of three parts F‐12 medium (Sigma‐Aldrich) and one part DMEM (Sigma‐Aldrich) supplemented with 5% FBS (Sigma‐Aldrich), 0.4 μg/ml hydrocortisone, 5 μg/ml insulin, 8.4 ng/ml cholera toxin, 24 μg/ml adenine, 10 ng/ml epidermal growth factor (EGF), 100 g/ml streptomycin, and 100 IU/ml penicillin.
The immortalized human keratinocytes cell line HaCaT (RRID:CVCL_0038) was purchased from Cosmo Bio. HaCaT cells were cultured at 37°C under 5% CO2 in DMEM (Sigma‐Aldrich) or calcium‐free DMEM (Sigma‐Aldrich) supplemented with 10% FBS (Sigma‐Aldrich), 100 g/ml streptomycin, and 100 IU/ml penicillin.
The human cervical squamous carcinoma cell line C33A (RRID:CVCL_1094), which was authenticated by STR profiling, was used in this study. C33A cells (HPV negative) were cultured at 37°C under 5% CO2 in DMEM (Sigma‐Aldrich) supplemented with 10% FBS (Sigma‐Aldrich), 100 g/ml streptomycin, and 100 IU/ml penicillin.
The immortalized human keratinocytes cell line HaCaT (RRID:CVCL_0038) was purchased from Cosmo Bio. HaCaT cells were cultured at 37°C under 5% CO2 in DMEM (Sigma‐Aldrich) or calcium‐free DMEM (Sigma‐Aldrich) supplemented with 10% FBS (Sigma‐Aldrich), 100 g/ml streptomycin, and 100 IU/ml penicillin.
The human cervical squamous carcinoma cell line C33A (RRID:CVCL_1094), which was authenticated by STR profiling, was used in this study. C33A cells (HPV negative) were cultured at 37°C under 5% CO2 in DMEM (Sigma‐Aldrich) supplemented with 10% FBS (Sigma‐Aldrich), 100 g/ml streptomycin, and 100 IU/ml penicillin.
5
Cytotoxicity Assay with HaCaT Cells
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6
Culturing Human Melanoma and Epidermal Cells
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Human immortalized epidermal cell line (HaCaT, #BNCC101683) was obtained from BeNa culture collection. A total of five human melanoma cell lines used in this study, including 451Lu cell line, A-375, VMM5A, SK-MEL-1 and G-361 cell lines. Only the first cell line was kindly provided by Dr. MeenhardHerlyn (Wistar Institute, PA, USA), the rest four ones were all purchased from the American Type Culture Collection (#CRL-1619, #CRL-3226, #HTB-67 and #CRL-1424™). HaCaT cells were cultured with Minimum Essential Medium (MEM, #56419C, Sigma-Aldrich, St. Louis, MO, USA), and all melanoma cell lines were incubated with Dulbecco’s modified eagle’s medium (DMEM, #D5030, Sigma-Aldrich). Both cell culture mediums contained 10% fetal bovine serum (FBS, #10099141, ThermoScientific, Hudson, NH, USA) and 1% penicillin-streptomycin, and then the cells were kept in a humidified incubator with 5% CO2 at 37 °C.
7
Cell Line Cultivation and Maintenance
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The normal human fibroblasts cell line BJ, colorectal cancer cell line DLD-1, squamous cell lung carcinoma cell line SK-MES-1, triple negative breast cancer cell line MDA-MB-231, and double positive breast cancer cell line MCF-7 were purchased from the American Type Culture Collection (ATCC) (Manassas, VA, USA) and the normal human keratinocyte HaCaT was acquired from the Cell Line Service of the German Cancer Research Centre in Heidelberg, Germany. BJ and MCF-7 were maintained in minimum essential medium Eagle—MEM (Sigma Aldrich, ST. Louis, MI, USA), MDA-MB-231, DLD-1, and SK-MES-1 in RPMI (Sigma Aldrich, St. Louis, MI, USA), and HaCaT in Dulbecco’s modified Eagle medium—DMEM with low glucose (Sigma Aldrich, St. Louis, MI, USA) [74 (link),75 (link)], all supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA), 1% glutamine (Gibco, Grand Island, NY, USA), and 1% penicillin-streptomycin (Gibco, Grand Island, NY, USA).
8
Cytotoxicity of EJ-AuNPs on HaCaT Cells
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Human epidermal keratinocytes (HaCaT; CLS GmbH, Eppelheim, Germany) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; GenDEPOT, Katy, TX, United States) containing 10% fetal bovine serum (FBS; GenDEPOT), 100 U penicillin, and 100 μg/ml streptomycin (GenDEPOT). The cells were then incubated in a humidified incubator with 5% carbon dioxide (CO2)/95% air. Cells (1 × 104 cells) were plated in a 96-well plate (SPL Life Sciences, Pocheon, Korea) and stabilized for 24 h. The dried EJ-AuNPs or EJ was diluted with serum-free medium (SFM) at concentrations of 25, 50, and 100 μg/ml. After the cells were washed twice with phosphate-buffered saline (PBS), diluted EJ-AuNPs solution was added to the cells and incubated for a further 24 h. To compare the cytotoxic effect of EJ-AuNPs, only SFM and commercial dexamethasone (20 μg/ml)-containing SFM were added to the cells. The cytotoxic effects of EJ-AuNPs against HaCaT cells were measured using a conventional 3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) solution (Sigma-Aldrich, St. Louis, MO, United States) and a live/dead cell staining kit (Thermo Fisher Scientific, Cambridge, MA, United States), according to the manufacturer’s instructions.
9
Standardized Cell Culture Protocols
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Human colorectal adenocarcinoma cell line HT-29 (ATCC, Manassas, VA, USA), HCT116 (a kind gift from Professor Sung Gu Han, Konkuk University), human breast cancer cell lines (MCF7 and MDA-MB231), hepatocellular carcinoma cells (HepG2), neuroblastoma cells (SH-SY5Y), and immortalized human keratinocytes (HaCaT) (ATCC, Manassas, VA, USA) were cultured in DMEM (HT29, MCF7, HepG2, SH-SY5Y, and HaCaT cell lines) or RPMI 1640 (HCT116 and MDA-MB231 cell lines) (Sigma-Aldrich, Saint Louis, MO, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific Ltd., Waltham, MA, USA), 100 U/mL penicillin and 100 mg/mL streptomycin (Gibco). Cells were retained at 37 °C in a humidified atmosphere of 5% CO2. Possible mycoplasma contaminations in all cell lines were tested using a BioMycoX® Mycoplasma PCR Detection Kit (Cat. No. CS-D-25) (Cellsafe, Yeongtong-gu, Suwon, Korea) and were authenticated using short tandem repeat (STR) profiling.
10
Culturing Mouse Fibroblasts and Human Keratinocytes
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A mouse embryo fibroblast cell line and a spontaneously immortalized human skin keratinocyte line (HaCaT) were purchased from ATCC and cultured as described. Briefly, fibroblasts were seeded and grown in Dulbecco’s modified Eagle’s medium (DMEM; Sigma, USA) with 10% fetal bovine serum (FBS) and a penicillin/streptomycin mixture under an atmosphere of 5% CO2 at 37 °C. HaCaT cells were seeded and grown in RPMI-1640 medium (Sigma, USA) containing 10% FBS and a penicillin/streptomycin mixture under an atmosphere of 5% CO2 at 37 °C. The cultures were passaged twice before the experiments.
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