Cnt 57 medium

Manufactured by CELLnTEC

Sourced in Switzerland

Cnt-57 medium is a cell culture medium formulated for the growth and maintenance of cell lines. It provides the necessary nutrients and growth factors required for cell proliferation and survival in vitro. The composition of Cnt-57 medium is designed to support the specific needs of a variety of cell types.

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CnT-57 (7 citations)

7 protocols using cnt 57 medium

Cell Culture and Genetic Manipulation

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A431 or HeLa (ATCC) cells were grown in Dulbecco’s modified essential medium (Invitrogen) containing 10% FBS (Atlanta Biologicals), 100 units/ml penicillin, and 100 µg/ml streptomycin (Invitrogen) at 37°C in 5% CO₂. For cells stably expressing KRT17 or SCR shRNA, the medium was supplemented with 0.5 µg/ml puromycin. Primary cultures of skin keratinocytes from 2-d-old mouse littermate pups that are either heterozygous (Krt17^+/−) or homozygous (Krt17^−/−) for a Krt17 null allele (McGowan et al., 2002 (link)) were isolated and cultured as described previously (Chung et al., 2012 (link)). Primary cultures of skin keratinocytes from ear lesions of age-matched Gli2^tg/+ mice that are either WT (Krt17^+/+), heterozygous (Krt17^+/−), or homozygous (Krt17^−/−) for the Krt17 allele were isolated by mincing ears into fine pieces and subjecting them to 0.25% Trypsin overnight. After vigorous vortexing, free keratinocytes were isolated as described previously (Chung et al., 2012 (link)) and cultured in Cnt-57 medium (CELLnTEC) before harvesting. For TPA stimulation, cells previously incubated for 18 h in 0.1% FBS-containing medium were treated with either DMSO (vehicle control) or 200 nM TPA for the indicated time periods.

Chung B.M., Arutyunov A., Ilagan E., Yao N., Wills-Karp M, & Coulombe P.A. (2015). Regulation of C-X-C chemokine gene expression by keratin 17 and hnRNP K in skin tumor keratinocytes. The Journal of Cell Biology, 208(5), 613-627.

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Isolation of Primary Mouse Fibroblasts and Keratinocytes

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Primary MEFs were isolated from 14.5-day-old Erbin^+/+ and Erbin^−/− FVB mouse embryos. After dissection, fetuses were transferred in sterile PBS, livers and heads were removed and discarded, and the remaining part of each fetus was teased into fine pieces. Tissues were broken up into a suspension by means of vigorous pipetting, and after a short sedimentation, the remaining cell suspension was grown in Dulbecco modified Eagle medium supplemented with 10% FCS and antibiotics.
Primary mouse epidermal keratinocytes were isolated from 19.5-day-old Erbin^+/+ and Erbin^−/− BALB/c mouse embryos. After dissection, fetuses were transferred in sterile PBS; heads, paws, and tails were removed and discarded; and back skins were removed and treated with Dispase (Gibco/Invitrogen, Carlsbad, Calif). The epidermis was separated from the dermis with forceps and incubated with a trypsin/versene mixture. The epidermis was cut into small pieces, and after pipetting vigorously up and down, the basal layer keratinocytes were separated out from the epidermis. The cell suspension was filtered, washed with medium, and grown in CnT-57 medium (CELLnTEC, Bern, Switzerland) containing a low concentration of bovine pituitary extract.

Polivka L., Hadj-Rabia S., Bal E., Leclerc-Mercier S., Madrange M., Hamel Y., Bonnet D., Mallet S., Lepidi H., Ovaert C., Barbet P., Dupont C., Neven B., Munnich A., Godsel L.M., Campeotto F., Weil R., Laplantine E., Marchetto S., Borg J.P., Weis W.I., Casanova J.L., Puel A., Green K.J., Bodemer C, & Smahi A. (2018). Epithelial barrier dysfunction in desmoglein-1 deficiency. The Journal of allergy and clinical immunology, 142(2), 702-706.e7.

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Keratinocyte Hydrolysate Response to IL-13

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A pool of 4 x 10⁴ primary human epidermal keratinocytes (CELLnTec, Bern, Switzerland) were seeded in a 48-well plate (Corning, VWR, Nyon, Switzerland) in CnT57 medium (CELLnTec, Bern, Switzerland) supplemented with 10% serum. When keratinocytes reached 90% confluence, cells were treated with the hydrolysate at 1 µg protein/mL. One hour after the addition of the ingredient, 50 ng/mL of interleukin-13 (IL-13) (Peprotech, London, UK) was added and incubated at 37 °C with 5% CO₂ for 48 h. An identical volume of medium was added to the non-IL-13 primed cultures.

Holvoet S., Nutten S., Dupuis L., Donnicola D., Bourdeau T., Hughes-Formella B., Simon D., Simon H.U., Carvalho R.S., Spergel J.M., Koletzko S, & Blanchard C. (2021). Partially Hydrolysed Whey-Based Infant Formula Improves Skin Barrier Function. Nutrients, 13(9), 3113.

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Isolation and Culture of Human Limbal Epithelial Cells

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Primary human limbal epithelial (HLE) cells were cultured in Cnt-57 medium provided by CELLnTEC Advanced Cell Systems (Bern, Switzerland). When used to HLE culture, this media is promoting a putative LESC phenotype. Sectioned corneo-scleral buttons or limbal rims were treated with a 1.2 U/ml dispase II solution (Sigma) for 2 h at 37 °C. Then, the epithelial cells were gently scraped by using a scalpel and following the limbal border in order to achieve an enriched LESC/progenitor population. The cells were then placed into a T-25 tissue culture flask (Nunc). The cultures were cultured at 37 °C and 5 % CO₂ in air; epithelial colonies emerged 3–5 days following isolation. The cells reached 80–90 % confluence in approximately 10 days and were always used at P0.

Domdey M., Kluth M.A., Maßlo C., Ganss C., Frank M.H., Frank N.Y., Coroneo M.T., Cursiefen C, & Notara M. (2022). Consecutive dosing of UVB irradiation induces loss of ABCB5 expression and activation of EMT and fibrosis proteins in limbal epithelial cells similar to pterygium epithelium. Stem Cell Research, 64, 102936.

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Primary Keratinocyte Culturing Protocol

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Human primary keratinocytes were used in this study including human primary neonatal foreskin keratinocytes (HPK) (Lonza, Basel, Switzerland), human primary adult foreskin keratinocytes single donor NUH49 and TB3 (HPEKas.05, CELLnTEC, Bern, Switzerland). The keratinocytes were cultured in supplemented CnT-57 medium (CELLnTEC, Bern, Switzerland). All cells used were tested and found free of mycoplasma.

Sunthamala N., Thierry F., Teissier S., Pientong C., Kongyingyoes B., Tangsiriwatthana T., Sangkomkamhang U, & Ekalaksananan T. (2014). E2 Proteins of High Risk Human Papillomaviruses Down-Modulate STING and IFN-κ Transcription in Keratinocytes. PLoS ONE, 9(3), e91473.

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Isolation of Human Limbal Epithelial Progenitors

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Primary human limbal epithelial (HLE) cells were cultured in a CNT-57 medium provided by CELLnTEC Advanced Cell Systems (Bern, Switzerland). Sectioned corneo–scleral buttons or limbal rims were treated with a 1.2 U/mL dispase II solution (Sigma) for 2 h at 37 °C. Then, the epithelial cells were gently scraped by using a scalpel and following the limbal border in order to achieve an enriched LESC/progenitor population. The cells were then placed into a T-25 tissue culture flask (Nunc). The cultures were cultured at 37 °C and 5% CO₂ in air; epithelial colonies emerged 3–5 days following isolation. Once the well was at approximately 80% confluent, fibroblasts were removed by using MACS depletion columns and Anti-Fibroblast MicroBeads human (Order no. 130-050-601, Miltenyi Biotec B.V. & Co. KG, Bergisch Gladbach, Germany).

Meshko B., Volatier T.L., Hadrian K., Deng S., Hou Y., Kluth M.A., Ganss C., Frank M.H., Frank N.Y., Ksander B., Cursiefen C, & Notara M. (2023). ABCB5+ Limbal Epithelial Stem Cells Inhibit Developmental but Promote Inflammatory (Lymph) Angiogenesis While Preventing Corneal Inflammation. Cells, 12(13), 1731.

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Isolation of Primary Human and Mouse Keratinocytes

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Primary normal human epidermal keratinocytes (NHEKs) isolated from neonatal foreskin (Lonza, Basel, Switzerland) were cultured in keratinocyte growth medium (Lonza). To isolate primary mouse keratinocytes (NMKs), neonatal WT mice on a C57BL/6J background (Clea, Tokyo, Japan) were sacrificed, and whole-skin sections were incubated with 0.25% trypsin EDTA (Life Technologies, Carlsbad, CA) for 1 hour at 37 C. After the epidermal layer was scraped and incubated with 10% fetal calf serum in phosphate-buffered saline (PBS; Life Technologies), primary NMKs were seeded on plastic with Cnt-57 medium supplemented with bovine pituitary extract (Cellntec, Bern, Switzerland). Cells up to the fourth passage were used.

Nishie W., Natsuga K., Iwata H., Izumi K., Ujiie H., Toyonaga E., Hata H., Nakamura H, & Shimizu H. (2015). Context-Dependent Regulation of Collagen XVII Ectodomain Shedding in Skin. The American journal of pathology, 185(5).

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