Human HaCaT keratinocytes (kindly provided by Dr. Fusenig), AMPK wild-type (WT) and knockout (KO) mouse embryonic fibroblast (MEF) cells,12 (link) ATG5 WT and KO MEF cells (kindly provided by Dr. Mizushima), and 143B WT and cytochrome b null (CYTB KO) cells (kindly provided by Dr. Chandel)13 (link), 14 were maintained in monolayer culture in 95% air/5% CO2 at 37 °C in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 units per mL penicillin, and 100 mg per mL streptomycin (Invitrogen, Carlsbad, California). Normal human epidermal keratinocytes (NHEK) cells were obtained from Clonetics (Lonza, Walkersville, MD) and cultured in KGM Gold BulletKit medium (Lonza, Walkersville, MD) according to the manufacturer's instructions. MEF and HaCaT cells were cultured for less than 20 passages. NHEK cells were cultured for less than 4 passages. No authentication was done.
Kgm gold bulletkit medium
Manufactured by Lonza
Sourced in United States, Japan
The KGM Gold BulletKit medium is a cell culture medium designed for the growth and maintenance of human keratinocytes. It provides a standardized and optimized formulation to support the proliferation and differentiation of keratinocytes in vitro.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Ker ct, by American Type Culture Collection (1 mentions)
Hek001, by American Type Culture Collection (1 mentions)
Keratinocyte serum free medium, by Thermo Fisher Scientific (1 mentions)
Isoproterenol hydrochloride, by Merck Group (1 mentions)
Medium 254, by Thermo Fisher Scientific (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Atlanta Biologicals (1 mentions)
Nunc microwell 96 well microplate, by Thermo Fisher Scientific (1 mentions)
Fgm 2 bulletkit medium, by Lonza (1 mentions)
Nhek ad cells, by Lonza (1 mentions)
5 protocols using kgm gold bulletkit medium
1
Cell Culture Protocols for Various Cell Lines
2
Culturing Human Keratinocyte Cell Lines
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The HEK001 (CRL-2404, ATCC) human keratinocyte cell line, which was obtained from an older human subject, was cultured in keratinocyte-serum free medium (Gibco, Carlsbad, CA, USA, 17005-042) supplemented with human recombinant EGF (5 ng/mL), gentamicin (10 mg/mL), and 2 mM L-glutamine. The Ker-CT (CRL-4048, ATCC) human neonatal keratinocyte cell line was cultured in KGM Gold BulletKit medium (Lonza, Walkersville, MD, USA, 00192060). These cell lines were maintained in a humidified incubator at 37 °C with 5% CO2. The protocols for each specific treatment are described in the figure legends.
3
Cell Culture Maintenance Protocols
4
Isolation and Culture of Normal Human Skin Cells
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Normal human skin cells were isolated from donated skin samples (1 adult female breast skin sample and 12 neonatal foreskin samples) obtained from Dr. George Xu at University of Pennsylvania and Dr. David M. Owens at Columbia University under protocols approved by the respective institute's Review Board. Cells were isolated as described previously.55 , 84 , 85 Normal human epidermal keratinocytes (NHEK) were cultured in KGM® Gold Bullet Kit medium (Lonza, #192060) supplemented with isoproterenol hydrochloride 10−6 M (Sigma‐Aldrich, #I6504). Fibroblasts were cultured in Dulbecco's Modified Eagle Medium (DMEM) (Gibco™ by Life Technologies, #11995) supplemented with 10% fetal bovine serum (FBS) (Atlanta Biologicals, #S115500H) and melanocytes were maintained in Medium 254 (Gibco™ by Life Technologies, #M254500), supplemented with Human Melanocyte Growth Supplement (HMGS) (Gibco™ by Life Technologies, #S0025). Fibroblasts and melanocytes were maintained in a humidified chamber at 37°C containing 5% CO2, and keratinocytes were maintained at 37°C and 7.5% CO2. Cells were sub‐cultured by treatment with 0.05% trypsin–EDTA (Gibco™ by Life Technologies, #25300‐054) when they reached approximately 80% confluency.
5
In Vitro Fibroblast and Keratinocyte Assays
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Adult normal human dermal fibroblasts, NHDF‐Ad cells (Lonza Japan Ltd, Tokyo, Japan, CC‐2511), and adult normal human epidermal keratinocytes, NHEK‐Ad cells (Lonza Japan Ltd, 00192627), were cultured in FGM™ − 2 BulletKit™ medium (Lonza Japan Ltd)12 and KGM‐Gold™ BulletKit™ medium (Lonza Japan Ltd),13 respectively. In total, 1 × 104 NHDF‐Ad cells were added to each well of Nunc™ MicroWell™ 96‐well microplates (Thermo Fisher Scientific K. K., Tokyo, Japan) before being pre‐cultured for 24 hours in the cytotoxicity assay. In total, 2 × 105 NHEK‐Ad cells were added to each well of Nunc™ MicroWell™ 12‐well microplates (Thermo Fisher Scientific K. K.) before being pre‐cultured for 20 hours in the scratch assay and for 24 hours in the live/dead assay. All cells were cultured in a humidified atmosphere containing 5% CO2 at 37°C.
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