PMKs were isolated from newborn 2- to 3-day-old C57Bl/6 mice, as described previously (Lewis et al., 2014 (link), Mardaryev et al., 2016 (link)). PMKs were grown in EMEM calcium-free medium (Lonza, Wolverhampton, UK) with supplements (0.05 mM calcium, 4% fetal bovine serum, 0.4 μg/ml hydrocortisone, 5 μg/ml insulin, 10 mg/ml epidermal growth factor (EGF), 10−10 M cholera toxin, 2 × 10−9 T3, 2 mM l -glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin) at 33 °C, 8% CO2 until 60–70% confluent. PMKs were transfected with 100 nM p63siRNA or control siRNA using Lipofectamin 2000 (Life Technologies).
Emem calcium free medium
Manufactured by Lonza
Sourced in United Kingdom
EMEM calcium-free medium is a cell culture medium formulation designed for in vitro studies. It provides a balanced salt solution and essential nutrients to support the growth and maintenance of cells in the absence of calcium ions.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamin 2000, by Thermo Fisher Scientific (2 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions)
70 m cell strainer, by BD (1 mentions)
Sca 1, by Beckman Coulter (1 mentions)
Cd49f, by Beckman Coulter (1 mentions)
Summit software, by Beckman Coulter (1 mentions)
Cd49f, by BD (1 mentions)
Moflo xdp cell sorter, by Beckman Coulter (1 mentions)
4 protocols using emem calcium free medium
1
Isolation and Transfection of Primary Murine Keratinocytes
2
miR-214 Regulation of Wnt Signaling in Primary Mouse Epidermal Keratinocytes
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PMEKs were prepared from newborn mice at P2–3. In brief, mouse skins were incubated in 0.25% trypsin at 4°C overnight. The epidermal portion was minced and filtered through a 70-µm cell strainer (BD Biosciences), which result in a single-cell suspension. PMEKs were grown in EMEM calcium-free medium (Lonza) supplemented with 0.05 mM calcium, at 33°C, 8% CO2 (Scientific Laboratory Suppliers, Hessle, UK) until 60–70% confluent. PMEKs were transfected with 200 nM of synthetic miR-214 inhibitor (anti-miR-214), miR-214 mimic (pro-miR-214), or miRNA negative controls (GE Healthcare), using Lipofectamine RNAiMax (Invitrogen). Cells were harvested 24 h after transfection and used for further analyses. To induce Wnt signaling, PMEKs were treated with 10 mM lithium chloride (Klein and Melton, 1996 (link)). To examine the regulatory effects of miR-214 on Wnt/β-Catenin signaling, PMEKs were treated with 10 mM LiCl for 2 h, followed by transfection of cells with 200 nM pro-miR-214 or miRNA negative controls for 4 h at 33°C, 8% CO2. Cells were then harvested 24 h after transfection.
3
Isolation and Transfection of Murine Epidermal Keratinocytes
4
Hair Follicle Stem Cell Isolation
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Skins from C57Bl/6 WT mice were harvested between ages 7 and 9 weeks. In brief, dissected and minced tissue was filtered using a 40-μm filter. The cells were centrifuged down at 300g for 10 minutes and resuspended in 1 ml of EMEM calcium-free medium (Lonza, Manchester, United Kingdom). Cells were stained with Ly-6A/E (SCA-1) (Thermo Fisher Scientific), CD34 (RAM34) (Thermo Fisher Scientific), and CD49f (BD Pharmingen, San Diego, CA) in a 2% BSA/PBS staining buffer rotating for 1 hour at 4 °C. Cells were then stained with secondary antibody allophycocyanin streptavidin (BioLegend, San Diego, CA) for 1 hour at 4 °C. CD34 + /CD49f High /SCA-1 HF-bulge SCs, CD34 /CD49f high /SCA-1 + basal SCs, and CD34 /CD49f Low /SCA-1 suprabasal KCs were sorted using a MoFlo-XDP cell sorter (Beckman Coulter, High Wycombe, United Kingdom), and data were analyzed using Summit software (Beckman Coulter). Sorted SCs were then used for subsequent colony-forming assays and RT-qPCR analysis.
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