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Epidermal growth factor 1 53

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Epidermal Growth Factor 1–53 is a recombinant human protein that corresponds to the first 53 amino acids of the epidermal growth factor (EGF) sequence. It is a widely used growth factor in cell culture applications.

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Bovine pituitary extract, by Thermo Fisher Scientific (5 mentions) Sytox blue dead cell stain, by Thermo Fisher Scientific (2 mentions)

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Human recombinant epidermal growth factor 1–53 (4 citations)

5 protocols using epidermal growth factor 1 53

1

Organotypic Skin Cultures with Dermal Fibroblasts

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Primary human keratinocytes were cultured in Keratinocyte Serum Free Medium supplemented with Epidermal Growth Factor 1–53 and Bovine Pituitary Extract (Life Technologies; 17005042). Generation of organotypic skin cultures were performed as described in Li and Sen, 2015. Briefly, ~500K control cells were seeded on devitalized human dermis and raised to an air/liquid interface to induce differentiation and stratification over the indicated number of days with culture changes every two days. Prior to seeding keratinocytes, either Matrigel was applied to the underside of the devitalized dermis or primary human dermal fibroblasts were centrifuged into the devitalized dermis. To evaluate the effect of oxygen levels on 3D skin cultures, FibHSEs were cultured as previously described and exposed to either normoxia (18–20% oxygen) or hypoxia (3% oxygen) at the air-liquid interface for 14 days. To measure changes from EGF supplementation, culture medium was switched to Keratinocyte Serum Free Medium supplemented with Bovine Pituitary Extract and variable concentrations of Epidermal Growth Factor 1–53 (Life Technologies; 17005042) after one week for one additional week of culturing.
Stabell A.R., Lee G.E., Jia Y., Wong K.N., Wang S., Ling J., Nguyen S.D., Sen G.L., Nie Q, & Atwood S.X. (2023). Single-cell transcriptomics of human-skin-equivalent organoids. Cell reports, 42(5), 112511.
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2

Organotypic Skin Cultures with Dermal Fibroblasts

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Primary human keratinocytes were cultured in Keratinocyte Serum Free Medium supplemented with Epidermal Growth Factor 1–53 and Bovine Pituitary Extract (Life Technologies; 17005042). Generation of organotypic skin cultures were performed as described in Li and Sen, 2015. Briefly, ~500K control cells were seeded on devitalized human dermis and raised to an air/liquid interface to induce differentiation and stratification over the indicated number of days with culture changes every two days. Prior to seeding keratinocytes, either Matrigel was applied to the underside of the devitalized dermis or primary human dermal fibroblasts were centrifuged into the devitalized dermis. To evaluate the effect of oxygen levels on 3D skin cultures, FibHSEs were cultured as previously described and exposed to either normoxia (18–20% oxygen) or hypoxia (3% oxygen) at the air-liquid interface for 14 days. To measure changes from EGF supplementation, culture medium was switched to Keratinocyte Serum Free Medium supplemented with Bovine Pituitary Extract and variable concentrations of Epidermal Growth Factor 1–53 (Life Technologies; 17005042) after one week for one additional week of culturing.
Carlino A., Toledo H., Skaleris D., DeLisio R., Weissbach H, & Brot N. (1992). Interactions of liver Grp78 and Escherichia coli recombinant Grp78 with ATP: multiple species and disaggregation.. Proceedings of the National Academy of Sciences of the United States of America, 89(6), 2081-2085.
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3

Organotypic Skin Culture from Primary Keratinocytes

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Primary human keratinocytes were cultured in Keratinocyte Serum Free Media supplemented with Epidermal Growth Factor 1-53 and Bovine Pituitary Extract (Life Technologies; 17005042). For generating organotypic skin cultures, ~500 K control or knockdown cells were seeded on devitalized human dermis and raised to an air/liquid interface in order to induce differentiation and stratification over the indicated number of days with culture changes every 2 days64 (link).
Wang S., Drummond M.L., Guerrero-Juarez C.F., Tarapore E., MacLean A.L., Stabell A.R., Wu S.C., Gutierrez G., That B.T., Benavente C.A., Nie Q, & Atwood S.X. (2020). Single cell transcriptomics of human epidermis identifies basal stem cell transition states. Nature Communications, 11, 4239.
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4

Neonatal Foreskin Primary Cell Isolation

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Discarded and de-identified neonatal foreskins were collected during routine circumcision from UC Irvine Medical Center (Orange, CA, US). The samples were either processed for histological staining, single cell RNA-sequencing, or primary culture. No personal information was collected for this study. For primary cell isolation, fat from discarded and de-identified neonatal foreskins were removed using forceps and scissors and incubated with dispase epidermis side up for 2 hours at 37°C. The epidermis was peeled from the dermis, cut into fine pieces, and incubated in 0.25% Trypsin-EDTA for 15 minutes at 37°C and quenched with chelated FBS. Cells were passed through a 40μm filter, centrifuged at 1500rpm for 5 minutes, and the pellet resuspended in Keratinocyte Serum Free Medium supplemented with Epidermal Growth Factor 1–53 and Bovine Pituitary Extract (Life Technologies; 17005042). Cells were either live/dead sorted using SYTOX Blue Dead Cell Stain (ThermoFisher; S34857) for single cell RNA-sequencing or incubated at 37°C for culture.
Stabell A.R., Lee G.E., Jia Y., Wong K.N., Wang S., Ling J., Nguyen S.D., Sen G.L., Nie Q, & Atwood S.X. (2023). Single-cell transcriptomics of human-skin-equivalent organoids. Cell reports, 42(5), 112511.
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5

Neonatal Foreskin Primary Cell Isolation

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Discarded and de-identified neonatal foreskins were collected during routine circumcision from UC Irvine Medical Center (Orange, CA, US). The samples were either processed for histological staining, single cell RNA-sequencing, or primary culture. No personal information was collected for this study. For primary cell isolation, fat from discarded and de-identified neonatal foreskins were removed using forceps and scissors and incubated with dispase epidermis side up for 2 hours at 37°C. The epidermis was peeled from the dermis, cut into fine pieces, and incubated in 0.25% Trypsin-EDTA for 15 minutes at 37°C and quenched with chelated FBS. Cells were passed through a 40μm filter, centrifuged at 1500rpm for 5 minutes, and the pellet resuspended in Keratinocyte Serum Free Medium supplemented with Epidermal Growth Factor 1–53 and Bovine Pituitary Extract (Life Technologies; 17005042). Cells were either live/dead sorted using SYTOX Blue Dead Cell Stain (ThermoFisher; S34857) for single cell RNA-sequencing or incubated at 37°C for culture.
Carlino A., Toledo H., Skaleris D., DeLisio R., Weissbach H, & Brot N. (1992). Interactions of liver Grp78 and Escherichia coli recombinant Grp78 with ATP: multiple species and disaggregation.. Proceedings of the National Academy of Sciences of the United States of America, 89(6), 2081-2085.
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