KGMTM Gold Keratinocyte Growth Medium BulletKitTM

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	Third passage Neonatal Human Epidermal Keratinocytes (NHEKs Lonza) were initially cultured in serum-free conditions in Lonza's KGM-Gold media (basal medium supplemented with bovine pituitary extract, human epidermal growth factor, bovine insulin, hydrocortisone, Gentamicin, Amphotericin-B, Epinephrine and Transferrin) in collagen coated (1∶100; Type 1 rat tail collagen (Sigma) : PBS) T25 flasks. The cells were cultured in a 37°C incubator with 5% CO2, with the media being changed every 2 days until approximately 80% confluent.

Protocol tips

Third passage Neonatal Human Epidermal Keratinocytes (NHEKs Lonza) were initially cultured in serum-free conditions in Lonza's KGM-Gold media (basal medium supplemented with bovine pituitary extract, human epidermal growth factor, bovine insulin, hydrocortisone, Gentamicin, Amphotericin-B, Epinephrine and Transferrin) in collagen coated (1∶100; Type 1 rat tail collagen (Sigma) : PBS) T25 flasks. The cells were cultured in a 37°C incubator with 5% CO2, with the media being changed every 2 days until approximately 80% confluent.

Publication protocol

Third passage Neonatal Human Epidermal Keratinocytes (NHEKs Lonza) were initially cultured in serum-free conditions in Lonza's KGM-Gold media (basal medium supplemented with bovine pituitary extract, human epidermal growth factor, bovine insulin, hydrocortisone, Gentamicin, Amphotericin-B, Epinephrine and Transferrin) in collagen coated (1∶100; Type 1 rat tail collagen (Sigma) : PBS) T25 flasks. The cells were cultured in a 37°C incubator with 5% CO2, with the media being changed every 2 days until approximately 80% confluent. The NHEK's were then lifted with 3∶1 PBS/trypsin-EDTA (0.25%), which was neutralised by adding KGM-Gold media with 10% FBS before cells were centrifuged at 200× g for 5 minutes and counted with a hemocytometer. Cells were seeded into either a collagen coated T25 flask containing KGM-Gold or a collagen coated T25 flask using the method of Rheinwald and Green [10] at a density of 100000 cells per flask. The Rheinwald and Green method involves culturing the NHEKs on a mitotically inactivated feeder layer (JT-3T3 cells) in complete FAD media (made from 3 parts Dulbecco's modified Eagle's medium (DMEM) to 1 part Ham's F12 medium (F12) supplemented with 1.8×10−4 M adenine, 100 IU/ml penicillin, 100 µg/ml streptomycin, 10% fetal bovine serum, 0.5 ug/ml hydrocortisone, 8.47 ng/ml cholera enterotoxin, epidermal 10 ng/ml growth factor and 5 µg/ml insulin.

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