KGMTM Gold Keratinocyte Growth Medium BulletKitTM

Mammalian cell culture media HEK

Experiment
Mammalian cell culture media HEK
Product
KGMTM Gold Keratinocyte Growth Medium BulletKitTM from Lonza
Manufacturer
Lonza

Protocol tips

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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Papers

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Paper title
Keratinocytes Propagated in Serum-Free, Feeder-Free Culture Conditions Fail to Form Stratified Epidermis in a Reconstituted Skin Model
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Manufacturer protocol

Download the product protocol from Lonza for KGMTM Gold Keratinocyte Growth Medium BulletKitTM below.

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