S0015

Manufactured by Thermo Fisher Scientific

Sourced in United States

The S0015 is a laboratory instrument designed for analytical testing and measurement applications. It is a compact and user-friendly device that provides accurate and reliable results. The core function of the S0015 is to perform specific analytical tasks, but a detailed description of its intended use would require more information about the specific application.

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Lab products found in correlation

8 protocols using s0015

Keratinocyte Culture and Inhibitor Assays

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Normal human keratinocytes (NHKs) were established from healthy adults as previously described(60 (link)) and grown in medium 154 CF (Thermofisher M154CF500) with human keratinocyte growth supplement (Thermofisher S0015). Inhibitors used included the MEK1/2 inhibitor PD98059 (Tocris 1213), the MEK5 inhibitor Bix02189 (Tocris 4842) and the IκBα phosphorylation inhibitor Bay 11–7085 (Tocris 1743) according to the manufecturer’s specifications. siRNA was introduced by electroporation using Lonza 4D-nucleofector following manufacturer’s instructions. Cytokine stimulations were performed as previously reported(61 (link)). qRT-PCR was performed on a 7900HT Fast Real-time PCR system (Applied Biosystems) with TaqMan Universal PCR Master Mix (ThermoFisher 4304437).

Liang Y., Xing X., Beamer M.A., Swindell W.R., Sarkar M.K., Roberts L.W., Voorhees J.J., Kahlenberg J.M., Harms P.W., Johnston A, & Gudjonsson J.E. (2016). STEAPs comprise a novel inflammatory nexus in pustular skin disorders. The Journal of allergy and clinical immunology, 139(4), 1217-1227.

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Cryopreserved Human Keratinocytes and Fibroblasts

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Cryopreserved human keratinocytes (HEKa) (C0055C, Thermo Fisher Scientific) and human fibroblasts (HFs), isolated by abdominoplasty and graciously provided by Pr. Poumay (UNamur, Belgium), were thawed at 37°C. HEKa were mixed with cold keratinocyte medium (KCM) corresponding to EpiLife medium (MEPI500CA, Thermo Fisher Scientific) containing 0.06 mM CaCl₂, HKGS (S0015, Thermo Fisher Scientific), and 1% P/S. HFs were mixed in a fibroblast culture medium (FCM) consisting of DMEM (BE12-604F, Lonza, Westburg, Netherlands) containing 10% FBS (10270–106, Thermo Fisher Scientific), 1% L-glutamine (BE17-605E, Lonza, Westburg, Netherlands), and 1% P/S. The cells were cultured in a cell culture (37°C, 5% CO₂) incubator, and the medium was changed every 2 days.

Maistriaux L., Foulon V., Fievé L., Xhema D., Evrard R., Manon J., Coyette M., Bouzin C., Poumay Y., Gianello P., Behets C, & Lengelé B. (2024). Reconstruction of the human nipple–areolar complex: a tissue engineering approach. Frontiers in Bioengineering and Biotechnology, 11, 1295075.

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Neonatal Foreskin Keratinocyte Culture

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Primary human epidermal keratinocytes isolated from neonatal foreskin (nHEK) were provided and characterized by ThermoFisher (C0015C). Cells were cultured and expanded in low calcium, antibiotics-free and serum-free EpiLife Medium (ThermoFisher MEPI500CA), supplemented with human keratinocyte growth supplement (ThermoFisher S0015), according to the manufacturer's instructions. The nHEK cells and the culture conditions were mycoplasma-free. Low-passage (p3–p4), exponentially growing nHEK cells were used for all the experiments. siRNAs were transfected with Lipofectamine RNAi-MAX (ThermoFisher); DNA was transfected with Lipofectamine LTX PLUS Reagent (ThermoFisher). All transfections were performed in EpiLife Medium, according to the manufacturer's instructions. Unless otherwise indicated, siYAP/TAZ is mix siYAP/TAZ #1. Sequences of all siRNAs are provided in Supplementary Data 2.

Totaro A., Castellan M., Battilana G., Zanconato F., Azzolin L., Giulitti S., Cordenonsi M, & Piccolo S. (2017). YAP/TAZ link cell mechanics to Notch signalling to control epidermal stem cell fate. Nature Communications, 8, 15206.

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Culturing Immortalized Keratinocytes and Fibroblasts

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THEKs from original line N/TERT-2G (31 (link)) (a gift from James Rheinwald, Brigham and Women’s Hospital, Boston, Massachusetts, USA) were grown in keratinocyte serum-free medium (KSFM) ordered as a kit (Thermo Fisher Scientific catalog 37010022) with supplements of 0.2 ng/mL human epidermal growth factor and 30 μg/mL bovine pituitary extract. Other additives included 0.31 mM CaCl₂, 100 U/mL penicillin, and 100 μg/mL streptomycin.
NHEKs were procured as described below and grown in Medium 154 with 0.07 mM CaCl₂ (Thermo Fisher Scientific catalog M154CF500), 1× human keratinocyte growth supplement (Thermo Fisher Scientific catalog S0015), and 1× gentamicin/amphotericin (Thermo Fisher Scientific catalog R01510).
J2-3T3 immortalized murine fibroblasts (a gift from Kathleen Green, Northwestern University, Chicago, Illinois, USA) were grown in complete DMEM (Thermo Fisher Scientific catalog 11965092) supplemented with 10% FBS (Hyclone, Thermo Fisher Scientific catalog SH3039603), 2 mM GlutaMAX (Thermo Fisher Scientific catalog 35050061), 100 U/mL penicillin, and 100 μg/mL streptomycin.
All cell lines were maintained at 37°C in 5% CO₂ in an air-jacketed, humidified incubator. Cells were grown on sterile cell culture dishes and passaged at subconfluence using 0.25% Trypsin-EDTA (Thermo Fisher Scientific catalog 15400054).

Zaver S.A., Sarkar M.K., Egolf S., Zou J., Tiwaa A., Capell B.C., Gudjonsson J.E, & Simpson C.L. (2023). Targeting SERCA2 in organotypic epidermis reveals MEK inhibition as a therapeutic strategy for Darier disease. JCI Insight, 8(18), e170739.

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Keratinocyte Cytokine Profiling for Psoriasis

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The procedures have been described previously (36 ). Briefly, we obtained 50 normal human keratinocytes from 50 different healthy adults. Keratinocytes were grown in 12 well plate in 154 CF medium (Thermo Fisher #M154CF500) with human keratinocyte growth supplement (Thermo Fisher #S0015). Keratinocytes were grown to confluency at which time the complete medium (with supplements) was replaced by basal 154 CF medium (without supplements). Cells were stimulated with cytokines (IL-4, IL-13, IFN-α, IFN-γ, TNF-α, IL-17A, R&D Systems) individually at 10 ng/ml concentration. After 8hrs cells were harvested and RNA was isolated using RNeasy Plus Mini kit (Qiagen # 74136). RNA was analyzed by RNA Nano Chips (Agilent Technologies) and sequenced (37 ). We extracted the top 1,000 genes with their baseline expression profiles showing the strongest correlations with future absolute PASI improvement in each of the three follow-up visits, and used hypergeometric test to compare against cytokine signatures to understand their molecular basis.

Tsoi L.C., Patrick M.T., Shuai S., Sarkar M.K., Chi S., Ruffino B., Billi A.C., Xing X., Uppala R., Zang C., Fullmer J., He Z., Maverakis E., Mehta N.N., White B.E., Getsios S., Helfrich Y., Voorhees J.J., Kahlenberg J.M., Weidinger S, & Gudjonsson J.E. (2021). Cytokine responses in non-lesional psoriatic skin as clinical predictor to anti-TNF agents. The Journal of allergy and clinical immunology, 149(2), 640-649.e5.

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Isolation and Culture of Human Urothelial Cells

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hUCs were collected according to methods reported previously [14 (link), 18 (link)]. Briefly, 100–1000 ml of urine was collected from donors, centrifuged at 1010 × g for 5 minutes, and washed with phosphate-buffered saline (PBS). The cells were maintained in 24-well plates coated with 0.1% gelatin (ES-006-B; Millipore, Germany) in RM1 medium (50% Renal Epithelial Cell Growth Medium (REGM) (CC-3190; Lonza, USA) and 44% Dulbecco's Modified Eagle Medium (DMEM) (SH30022; HyClone, USA) supplemented with 5% fetal bovine serum (FBS) (P30-3302; PAN Biotech, Germany), 0.5% nonessential amino acids (NEAA) (11140050; Gibco, USA), 0.5% GlutaMax (35050-061; Gibco, USA)) and 1 × Primocin (ant-pm-2; InvivoGen, USA); 0.25% trypsin-EDTA (25200072; Gibco, USA) was used for dissociation of primary hUCs. RM1 or RM2 (82% DMEM (SH30022; HyClone, USA) supplemented with 5% FBS, 1% human keratinocyte growth supplement (HKGS) (S-001-5; Gibco, USA), 1% NEAA, and 1% GlutaMax) was used for hUC culture.
The HN4 hESC line was obtained from the Chinese Academy of Sciences, and both HN4 and hiPSCs were maintained in the hESC medium BioCISO (BC-PM0001; BIOCARE Biotech, China) in plates coated with Matrigel (354277; Corning, USA).

Wang L., Chen Y., Guan C., Zhao Z., Li Q., Yang J., Mo J., Wang B., Wu W., Yang X., Song L, & Li J. (2017). Using low-risk factors to generate non-integrated human induced pluripotent stem cells from urine-derived cells. Stem Cell Research & Therapy, 8, 245.

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Primary Mouse Keratinocyte Isolation and Culture

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Primary mouse keratinocytes and hair follicle buds were isolated from newborn pups as described previously (23 (link)) and cultured in modified Eagle’s medium (S-MEM, Gibco 98-0216DJ), 7% Chelex-treated FCS (Gemini Bio Products), and 0.05 mmol/L calcium unless otherwise indicated. MyD88-, EGFR-, and IκBζ-deficient keratinocytes were isolated from pups obtained from respective heterozygous breeding pairs (24 (link)). LSL-Hras^G12D mice were purchased from The Jackson Laboratory (stock #009046). TPA (Tocris) was diluted in DMSO. IL17A (R&D Systems, 421-ML-025), IL22 (R&D Systems, 582-ML-010), IL1ra (5 μg/mL, IL1ra or anakinra; Division of Veterinary Resources, NIH, Bethesda, MD) were diluted in culture medium and added to cell culture medium as indicated before cell harvesting. Neonatal human epidermal primary keratinocytes (Gibco, C-001-5C) were cultured in KSF media supplemented with human keratinocyte growth supplement (Gibco, S-001-5).

Cataisson C., Salcedo R., Michalowski A.M., Klosterman M., Naik S., Li L., Pan M.J., Sweet A., Chen J.Q., Kostecka L.G., Karwan M., Smith L., Dai R.M., Stewart C.A., Lyakh L., Hsieh W.T., Khan A., Yang H., Lee M., Trinchieri G, & Yuspa S.H. (2019). T-Cell Deletion of MyD88 Connects IL17 and IκBζ to RAS Oncogenesis. Molecular cancer research : MCR, 17(8), 1759-1773.

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Culturing Primary Human Keratinocytes

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Human primary keratinocytes all came from healthy female donors, 24–56 years of age (CellSystems GmbH, Troisdorf, Germany). They were maintained in EpiLife (Gibco, MEPI500CA) supplemented with HKGS (Gibco, S-001-5), at 37 °C in a 5% CO₂ humidified incubator.

Smiljanic S., Messaraa C., Lafon-Kolb V., Hrapovic N., Amini N., Osterlund C, & Visdal-Johnsen L. (2022). Betula alba Bark Extract and Empetrum nigrum Fruit Juice, a Natural Alternative to Niacinamide for Skin Barrier Benefits. International Journal of Molecular Sciences, 23(20), 12507.

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