Human keratinocyte growth supplement

Manufactured by Thermo Fisher Scientific

Sourced in United States, United Kingdom

The human keratinocyte growth supplement is a cell culture medium component that supports the growth and proliferation of human keratinocytes in vitro. It provides necessary growth factors and nutrients to maintain the viability and healthy growth of keratinocyte cultures.

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Growth supplement (20 citations) Human Keratinocyte Growth Supplement (HKGS (6 citations) Keratinocyte growth supplement (3 citations) Human Keratinocyte Growth Supplement Kit (2 citations) Human keratinocyte growth Supplement S7 (2 citations)

128 protocols using human keratinocyte growth supplement

Immortalized Keratinocytes for HPV Research

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Primary human foreskin keratinocytes (HFKs) were isolated from neonatal human foreskins. HFKs were grown in EpiLife medium (Gibco, Billings, MT, USA) supplemented with calcium chloride (Gibco), human keratinocyte growth supplement (Gibco), and penicillin-streptomycin (Caisson, North Logan, UT, USA) or Keratinocyte Growth Medium 2 (Promocell, Heidelberg, Germany), Supplement Mix (Promocell), and penicillin-streptomycin (Caisson). hTERT human foreskin keratinocytes (hTERT HFKs), provided by Michael Underbrink (University of Texas Medical Branch, Galveston, TX, USA), are immortalized keratinocytes that constituently express telomerase (hTERT). hTERT HFKs were grown in EpiLife medium (Gibco) supplemented with calcium chloride (Gibco), human keratinocyte growth supplement (Gibco), and penicillin-streptomycin (Caisson). Multiple passages were used throughout these experiments for both cell lines with hTERT HFK passaging ranging from 15–80 and primary HFKs passaging ranging from 9–11. hTERT HFKs and primary HFKs both expressed the control vector (LXSN) and β-HPV 8E6; hTERT HFKs expressed HA-tagged β-HPV 8E6. In total, one primary HFK and one hTERT HFK cell line (each from separate donors) was used in these experiments.

Snow J.A., Murthy V., Dacus D., Hu C, & Wallace N.A. (2019). β-HPV 8E6 Attenuates ATM and ATR Signaling in Response to UV Damage. Pathogens, 8(4), 267.

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Culturing Human Skin Cell Lines

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All cells were cultured at 37 °C in 5% CO 2 . Primary human dermal fibroblasts were previously described (Palmetshofer et al, 1995) . Fibroblasts and HaCaT cells were cultured in DMEM supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 U ml -1 penicillin, and 100 μg ml -1 streptomycin. Primary normal human keratinocyte was cultured as previously described (Pincelli et al., 1994) . Skin specimens obtained from repeat Caesarean section deliveries and circumcisions were used for cultures and grown in Epilife medium (Gibco BRL, Gaithersburg, MD) with human keratinocyte growth supplement (Gibco). Cells at passage 3-10 were used for experiments. HEKn cells were from Gibco and maintained according to the manufacturer's instructions in Epilife medium with human keratinocyte growth supplement. Cells at passage 3-6 were used for experiments. HaCaT/HEKn cells stably expressing RIP3 were created from cells infected with pLX303-hRIP3 lentiviral plasmid.

Kim S.K., Kim W.J., Yoon J.H., Ji J.H., Morgan M.J., Cho H., Kim Y.C, & Kim Y.S. (2015). Upregulated RIP3 Expression Potentiates MLKL Phosphorylation-Mediated Programmed Necrosis in Toxic Epidermal Necrolysis. The Journal of investigative dermatology, 135(8).

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Isolation and Conditioning of Skin Cells

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Keratinocytes and fibroblasts were isolated from normal human juvenile foreskins as described in Ref. [19 (link)] and under an approved protocol by the Committee on Human Research, University of California San Francisco and Veteran Affairs Medical Center, San Francisco.
Keratinocytes were grown in medium 154 supplemented with Human Keratinocyte Growth Supplement, 0.07 mmol/L Ca²⁺ (all Gibco) and 100 U/mL penicillin and 100 μg/mL streptomycin (Corning, Union City, CA). Fibroblasts were grown in DMEM (Gibco) supplemented with 10% fetal bovine serum (Gemini Bio-Products, West Sacramento, CA) and 100 U/mL penicillin and 100 μg/mL streptomycin (Corning).
Keratinocytes (2 × 10⁴ cell/cm²) and fibroblasts (5 × 10⁴ cell/cm²) were grown in their respective culture media and treated with 108 HKSA/mL for 4 hours. After three washes, media were refreshed. Keratinocyte-conditioned medium (K-CM) and fibroblast-conditioned medium (F-CM) were collected 24 hours later, centrifuged at 200 g for 5 minutes and filtered on 0.22 μm PVDF filter membrane (EMD Millipore, Temecula, CA). fetal bovine serum and calcium concentrations were adjusted to 2% and 1.8 mmol/L, respectively. Conditioned media were aliquoted, labeled as Control or HKSA (from cells without/with HKSA treatment respectively) and stored at −80°C.

Mainzer C., Packard T., Bordes S., Closs B., Greene W.C., Elias P.M, & Uchida Y. (2019). Tissue microenvironment initiates an immune response to structural components of Staphylococcus aureus. Experimental dermatology, 28(2), 161-168.

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Immortalized Human Foreskin Keratinocyte Culture

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Immortalized human foreskin keratinocytes (HFK), provided by Michael Underbrink (University of Texas Medical Branch, Galveston, TX, USA), were grown in EpiLife medium (Gibco, Gaithersburg, MD, USA), supplemented with 60 µM calcium chloride (Gibco), human keratinocyte growth supplement (Gibco), and 1% penicillin-streptomycin (Caisson, Smithfield, UT, USA). U2OS and HCT116 cells were maintained in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin. Zeocin (Alfa Aesar, Ward Hill, MA, USA) and H2O2 were used to induce DSBs. NU7441 (Selleckchem) was used to inhibit DNA-PKcs phosphorylation. KU55933 (Selleckchem, Houston, TX, USA) was used to inhibit ATM kinase activity.

Hu C., Bugbee T., Gamez M, & Wallace N.A. (2020). Beta Human Papillomavirus 8E6 Attenuates Non-Homologous End Joining by Hindering DNA-PKcs Activity. Cancers, 12(9), 2356.

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Keratinocyte Differentiation with Rottlerin

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NHEKs were established from skin biopsy samples incubated with dispase, as described earlier [25 (link)]. Briefly, detached keratinocytes without contamination were seeded onto flasks at a density of 5000 cells/cm² and maintained in Epilife Medium (Gibco, USA) containing human keratinocyte growth supplement (Gibco, USA) with media refreshed every 48–72 hours. The NHEKs were cultured at 37°C and 5% CO₂ in a humid atmosphere. Experiments were performed at passage 2–3. Keratinocytes were cultured in proliferation (0.03mM Cacl2 for 72h) or in differentiation conditions (1.2 mM Cacl2 for 48h). Keratinocytes cultured in 1.2mM Cacl2 medium were treated with media containing 0μM, 1μM, 5μM, 10μM rottlerin for the final 24 h (for a total of 72 hours of exposure to Ca2+).

Min M., Yan B.X., Wang P., Landeck L., Chen J.Q., Li W., Cai S.Q., Zheng M, & Man X.Y. (2017). Rottlerin as a therapeutic approach in psoriasis: Evidence from in vitro and in vivo studies. PLoS ONE, 12(12), e0190051.

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HPV16 E7 Gene Cloning and Expression

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HPV16-containing plasmid DNA (ATCC:45113D) was used as the template for PCR amplification and cloning of the HPV16 E7 gene into vector pEGFP-C1 following protocols described previously. NHEKs (ScienCell, Carlsbad, CA, United States) were cultured in 6-well plates using EpiLife culture medium (Gibco, United States) with 10% fetal bovine serum (Gibco, United States) and Human Keratinocyte Growth Supplement (Gibco, United States). NHEKs at 80% confluence were transfected with pEGFP-16E7 and pEGFP-C1 using Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA, United States). Six hours after Transfection, culture medium was replaced with fresh culture medium.

Hua C., Zhu J., Zhang B., Sun S., Song Y., van der Veen S, & Cheng H. (2020). Digital RNA Sequencing of Human Epidermal Keratinocytes Carrying Human Papillomavirus Type 16 E7. Frontiers in Genetics, 11, 819.

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Chemical Agents for Melanocyte and Keratinocyte Assays

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BCI-215, a chemical produced from BCI by medicinal chemistry [36 (link),37 (link),38 (link)], was purchased from MCE (MedChemExpress, Monmouth Junction, NJ, USA) and prepared as a solution in DMSO. Kojic acid, cholera toxin (CT), and 12-O-tetradecanoylphorbol-13-acetate (TPA) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Dulbecco’s Modified Eagle Medium (DMEM), Dulbecco’s phosphate-buffered saline (DPBS), and fetal bovine serum (FBS) were purchased from WelGENE (Daegu, South Korea). Medium 254, EpiLife™ Medium, Human Melanocyte Growth Supplement (HMGS), Human Keratinocyte Growth Supplement (HKGS), Antibiotic-Antimycotic (AA), and trypsin-EDTA were purchased from Gibco (Grand Island, NY, USA). Medium 254 (Gibco) was obtained from Cascade Biologics (Portland, OR, USA).

Lee J.H., Choi M.E., An H., Moon J.W., Yeo H.J., Song Y, & Chang S.E. (2022). BCI-215, a Dual-Specificity Phosphatase Inhibitor, Reduces UVB-Induced Pigmentation in Human Skin by Activating Mitogen-Activated Protein Kinase Pathways. Molecules, 27(17), 5449.

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Cell Culture and Treatment Protocols

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HEKs cells (catalog no. C‐005‐5C; Gibco) were cultured in EpiLife growth medium (catalog no. MEPI500CA; Gibco) supplemented with human keratinocyte growth supplement (catalog no. S‐001‐K; Gibco). Cells were seeded on collagen type IV (catalog no. C5533; Sigma‐Aldrich) precoated plates. Human SCC cells (A431; ATCC, VA) were cultured in Dulbecco's modified Eagle's medium (catalog no.11995‐065; Gibco) supplemented with 10% fetal bovine serum (catalog no. 098105; Multicel). PS‐1145 dihydrochloride (catalog no. P6624; Sigma‐Aldrich) was dissolved in dimethyl sulfoxide (DMSO; catalog no. D5879; Sigma‐Aldrich) and diluted at least 1000‐fold in culture medium before treatment. Tumor necrosis factor (TNF; catalog no.210‐TA‐020; R&D Systems) was dissolved in phosphate‐buffered saline (PBS) containing 0.1% bovine serum albumin (BSA; catalog no. A3095; Sigma‐Aldrich). FSL1 (Pam2CGDPKHPKSF, catalog no. tlr‐fsl; InvivoGen).

Altonsy M.O., Kurwa H.A., Lauzon G.J., Amrein M., Gerber A.N., Almishri W, & Mydlarski P.R. (2020). Corynebacterium tuberculostearicum, a human skin colonizer, induces the canonical nuclear factor‐κB inflammatory signaling pathway in human skin cells. Immunity, Inflammation and Disease, 8(1), 62-79.

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Plasmid Transfection of Keratinocytes

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Normal human epidermal keratinocytes (NHEKs) were cultured in EpiLife culture medium (Gibco, USA) with 10% fetal bovine serum (Gibco, USA) and Human Keratinocyte Growth Supplement (Gibco, USA). Siha cells were cultured in D MEM (Gibco, USA) containing 10% fetal bovine serum (Gibco, USA). C33A cells were cultured in MEM (Gibco, USA) with 10% fetal bovine serum (Gibco, USA) and 1% Penicillin-Streptomycin Solution (Gibco, USA). All cells were cultured at 37°C in the presence of 5% CO2. Cells at 80% confluence were transfected with GFP-LC3 or mCherry-GFP-LC3 plasmid using Lipofectamine 3000 reagent (Invitrogen, USA). The culture medium was replaced 6 h after transfection and the cells were cultured for another 48 h before analysis.

Hua C., Zheng Q., Zhu J., Chen S., Song Y., van der Veen S, & Cheng H. (2022). Human Papillomavirus Type 16 Early Protein E7 Activates Autophagy through Inhibition of Dual-Specificity Phosphatase 5. Oxidative Medicine and Cellular Longevity, 2022, 1863098.

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Culturing Human Keratinocyte Cell Lines

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HaCaT (Cell Lines Service – CLS, Germany) were cultured with Dulbecco’s modified Eagle medium (Gibco, Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (Sigma Aldrich) and 1% penicillin-streptomycin (Gibco, Thermo Fisher Scientific) at 5% CO₂ and 37°C. To passage and perform experiments, cells were detached with 10 min incubation with 5 mM EDTA solution in PBS followed by the incubation with 0.05% trypsin/EDTA (Gibco, Thermo Fisher Scientific). hKC cells were kindly provided by Dr Rudolf Leube. The cells were grown at 37°C and 5% CO₂ in EpiLife medium with human keratinocyte growth supplement (Gibco, Grand Island, NY) and penicillin/streptomycin. Cells were detached using accutase (Sigma) for 5 min at 37°C after incubation with 5 mM EDTA solution in PBS and resuspended in trypsin neutralizing solution (Gibco, Thermo Fisher Scientific) before use. Primary human keratinocytes (nHEK, CellSystems) were cultivated according to the manufacturer’s instructions for a maximum of three passages. Cell lines and primary cells were routinely tested negative for mycoplasma infection.

Di Russo J., Young J.L., Wegner J.W., Steins T., Kessler H, & Spatz J.P. (2021). Integrin α5β1 nano-presentation regulates collective keratinocyte migration independent of substrate rigidity. eLife, 10, e69861.

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