Mouse skin keratinocytes were isolated from newborn mice as described previously [61 (link)]. Cells were cultured in EMEM containing 0.05 mM CaCl2, 9% chFBS, penicilln 100 units/mL, streptomycin sulfate 100 U/mL, 10 ng/mL mouse epidermal growth factor (Thermo Fisher Scientific), and 50% filtered mouse fibroblast conditioned medium which was collected from culture of newborn mouse skin fibroblasts using DMEN medium supplemented with 10% chFBS.
Mouse epidermal growth factor
Manufactured by Thermo Fisher Scientific
Sourced in United States
Mouse epidermal growth factor is a recombinant protein that stimulates the proliferation and differentiation of various cell types. It is a key regulator of cell growth, survival, and migration.
Automatically generated - may contain errors
Lab products found in correlation
B27 supplement, by Thermo Fisher Scientific (6 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (5 mentions)
Glutamax, by Thermo Fisher Scientific (4 mentions)
Hepes, by Thermo Fisher Scientific (4 mentions)
N acetylcysteine, by Merck Group (4 mentions)
Pyruvate, by Avantor (4 mentions)
L glutamine, by Thermo Fisher Scientific (4 mentions)
Hydrocortisone, by Merck Group (3 mentions)
N2 supplement, by Thermo Fisher Scientific (3 mentions)
Penicillin, by Avantor (3 mentions)
Mouse leukemia inhibitory factor, by Santa Cruz Biotechnology (3 mentions)
Mouse basic fibroblast growth factor, by Thermo Fisher Scientific (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Matrigel, by Corning (2 mentions)
Alpha minimum essential medium, by Thermo Fisher Scientific (2 mentions)
Mouse glial cell line derived neurotrophic factor, by Thermo Fisher Scientific (2 mentions)
β mercaptoethanol, by Merck Group (2 mentions)
25 protocols using mouse epidermal growth factor
1
Isolation and Culture of Mouse Skin Keratinocytes
2
Embryonic Neural Stem Cell Isolation
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Mouse embryos were collected at E15.5 from timed pregnant dams. To obtain stem cell cultures from the developing V-SVZ, a “top-down” dissection approach was employed. Briefly, the heads were separated from the body, the two brain hemispheres were pulled back to either side to reveal the developing cortex which was collected as dorsal NSCs. The developing ganglionic eminences were collected as ventral NSCs. The collected tissue was minced and incubated at 37°C, 5% CO2 with 0.25% trypsin-EDTA solution for 20 minutes while rocking. The tissue was then gently dissociated via trituration with a pipette and the cells were cultured and maintained in media specific to embryonic neural stem cells as described in Moghadam et al., 2018 (link): Neurobasal media (ThermoFisher, 21103049); 1X B27 supplement without vitamin A (ThermoFisher, 12587010); 20 ng/mL mouse epidermal growth factor (ThermoFisher, 53003018); 10 ng/mL mouse basic fibroblast growth factor (ThermoFisher, PMG0035); 1 U/mL heparin (Sigma, 9041-08-01); 1X GlutaMax (ThermoFisher, 35050061); 1X modified Eagle’s medium non-essential amino acids (11140050); 0.1 mM β-mercaptoethanol; 10 μg/mL gentamicin. Cells were fed every 2–3 days and passaged upon reaching confluence.
3
Culturing Prostate Cancer Cell Lines
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The following CaP cell lines were used; LNCaP, 22Rv1, MDA-PCa-2B and PC3 (ATCC, Manassas, VA, USA). LNCaP, 22Rv1, and PC3 were cultured in RPMI1640 media (Invitrogen/GIBCO, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS, Omega Scientific, Inc., Tarzana, CA, USA), 2 mM L-glutamine, and 100 U/mL penicillin-100 μg/mL streptomycin (Invitrogen/GIBCO, Carlsbad, CA, USA). MDA-PCa-2B were cultured in F-12K media supplemented with 20% FBS (Omega Scientific, Inc., Tarzana, CA, USA), 2 mM L-glutamine, and 100 U/mL penicillin-100 μg/mL streptomycin (Invitrogen/GIBCO, Carlsbad, CA, USA), 25 ng/mL cholera toxin, 100 pg/mL hydrocortisone (Sigma-Aldrich, St. Louis, MO, USA), 10 ng/mL mouse Epidermal Growth Factor, 0.005 mM phosphoethanolamine, 45 nM sodium selenite, and 0.005 mg/mL human recombinant insulin (Thermofisher, Waltham, MA, USA). Cells were kept at 37 °C in a humidified environment of 5% CO2 in air.
4
Organoid Culture of Mouse Pancreatic Cells
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The mouse pancreatic tissues from KC/tdPd mice treated with the GW diet (50 mg/kg) or control diet for 3 days were digested with digestion buffer (1 mg/ml collagenase IV [Sigma-Aldrich] and 10 U/ml DNase I [Sigma-Aldrich] in DMEM) at 37 °C for 30 min, and the pancreatic epithelial cells were harvested, counted, and embedded in Matrigel (Corning). The Matrigel was topped with organoid culture medium comprising advanced DMEM/F-12 supplemented with penicillin–streptomycin, 2 mM GlutaMAX, 10 mM HEPES (all from Thermo Fisher Scientific), mouse recombinant Wnt-3A at 100 ng/ml (MilliporeSigma), mouse epidermal growth factor at 50 ng/ml (Invitrogen), mouse Noggin at 100 ng/ml (PeproTech), human R-spondin-1 at 1 µg/ml (PeproTech), N-acetyl-L-cysteine at 1 mM (Sigma-Aldrich), 1× N-2 supplement (Thermo Fisher Scientific), 1× B-27 supplement (Thermo Fisher Scientific), and Y-27632 at 10 µM (Fisher Scientific). PPARδ agonist GW (1 μM; Sigma-Aldrich) was added to organoid cultures for the mice treated with the GW diet. Organoids were imaged on days 1, 3, and 6 of culture unless otherwise specified. The conditioned culture media were harvested and passed through a 0.45-µm filter to remove the cell debris before being used for further studies.
5
Isolation and Culture of Intestinal Crypts
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Small intestines were removed, washed with ice-cold PBS, and cut into 3-cm–long pieces that were opened longitudinally. The villi were scraped off with a coverslip and the remaining tissue fragments were washed with PBS. Afterward, they were incubated in 1 mmol/L EDTA/PBS solution for 30 minutes at 4°C on a tube roller and transferred to 5 mmol/L EDTA/PBS for 1 hour at 4°C to enrich for small intestinal crypts. The crypt-containing solution was filtered through a 70-μm cell strainer, the crypts were counted, and centrifuged at 300 × g for 5 minutes at 4°C. The crypt-containing pellet was resuspended in a Matrigel matrix (Corning, Kaiserslautern, Germany) and seeded into a prewarmed 48-well plate. Matrigel was allowed to polymerize for 15 minutes at 37°C and the crypts were overlayed with Advanced Dulbecco’s modified Eagle medium/F12 supplemented with 1% Glutamax, 1% 1 mol/L HEPES, and 1% penicillin/ streptomycin, containing 1× N2, 1× B27 supplement (both from Invitrogen, Waltham, MA), 1.25 mmol/L n-acetylcysteine (Sigma-Aldrich), 0.05 μg/mL mouse epidermal growth factor (Invitrogen), 0.1 μg/mL murine Noggin (Peprotech, Hamburg, Germany), and 1 μg/mL recombinant human R-Spondin 1 (R&D Systems, Minneapolis, MN). The medium was changed every 3 days and the development was recorded with the EVOS FL Cell Imaging System (Thermo Scientific, Waltham, MA).
6
Feeder-Dependent Culture of Mouse Fetal Germ Stem Cells
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The FGSC line was established from mice as described in our previous reports [2 (link),37 (link)]. The mouse FGSC line was cultured in vitro according to previously described conditions [4 (link),38 (link)]. FGSCs were cultured in Minimum Essential Medium Alpha (Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (Life Technologies, Carlsbad, CA, USA), 2 mML-glutamine (Amresco, Radnor, PA, USA), 30 mg/mL pyruvate (Amresco), 1 mM nonessential amino acids (Invitrogen Life Sciences, CA, USA), 6 mg/mL penicillin (Amresco), 10 ng/mL mouse basic fibroblast growth factor (PeproTech, London, UK), 10 ng/mL mouse glial cell line-derived neurotrophic factor (PeproTech, NJ, USA), 20 ng/mL mouse epidermal growth factor (PeproTech), 10 ng/mL mouse leukemia inhibitory factor (Santa Cruz Biotechnology), and 50 mM β-mercaptoethanol (Sigma Chemical Co., St. Louis, MO, USA). The SIM-6-thiogunaniaoualiain (STO) cell line (ATCC, Manassas, VA, USA) served as the feeder to culture FGSCs. Cells were passaged every 5 days.
7
Culturing Fetal Germ Stem Cells
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In this study, we used the FGSC line, which was described in previous reports [8 (link), 11 (link), 12 (link)]. In brief, the FGSCs were cultured in minimum essential medium alpha (Invitrogen, Carlsbad, CA, USA), which was supplemented with 10% fetal bovine serum (Life Technologies, Carlsbad, CA, USA), 2 mM l -glutamine (Amresco, Radnor, PA, USA), 30 μg/mL pyruvate (Amresco), 1 mM nonessential amino acids (Invitrogen Life Sciences, CA, USA), 6 mg/mL penicillin (Amresco), 10 ng/mL mouse basic fibroblast growth factor (PeproTech, London, UK), 10 ng/mL mouse glial cell line-derived neurotrophic factor (PeproTech, NJ, USA), 20 ng/mL mouse epidermal growth factor (PeproTech), 10 ng/mL mouse leukemia inhibitory factor (Santa Cruz Biotechnology), and 50 mM β-mercaptoethanol (Sigma Chemical Co., St. Louis, MO, USA). SIM-6-thiogunaniaoualiain cells (ATCC, Manassas, VA, USA) served as the feeder to culture FGSCs, and were passaged every 4 days. FGSCs were identified by RT-PCR and immunofluorescence (Additional file 1 : Figure S2). We used a hemocytometer to count the cell number per well.
8
Oocyte Maturation in Transwell Culture
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The single follicles were cultured on transwell-COL membranes with medium, α-MEM supplemented with 5% FBS, 2% polyvinylpyrrolidone (Sigma), 2 mM L-glutamine, 200 μM ascorbic acid, 50 mMβ-mercaptoethanol, 30 mg/l penicillin, 20 ng/ml mouse epidermal growth factor (Pepro Tech), 75 mg/l streptomycin, 30 mg/ml pyruvate (Amresco), 0.2 IU/ml follicle-stimulating hormone (FSH; MSD), 20 ng /ml BMP15, and 20 ng/ml GDF9 (R&D Systems). At 2 days of culture, the culture medium was changed to medium without BMP15 and GDF9, and then follicles were incubated in 0.1% Type IV Collagenase (Invitrogen) for 5 min. After washing with α-MEM supplemented with 5% FBS several times, the follicles were cultured in medium without BMP15 and GDF9. After 14 days of culture, cumulus-oocyte complexes grown on the membrane were picked up by a fine glass capillary.
9
Culturing of Cell Lines for FcRn Studies
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HEK293E cells (ATCC, Manassas, VA, USA) and the J558L murine myeloma cell line stably producing NIP-specific hIgG1-IHH were cultured in RPMI 1640 (Sigma-Aldrich) supplemented with 10% heat-inactivated fetal calf serum (FCS) (Sigma-Aldrich), 2 mM l -glutamine, 25 μg/ml streptomycin, and 25 U/ml penicillin (all from Bio Whittaker). The parental human microvascular endothelial cell line (WT HMEC1) and HMEC1 stably expressing HA-hFcRn-EGFP (HMEC1-hFcRn)38 (link) were grown in MCDB 131 medium (Gibco) supplemented with 10% heat-inactivated FCS, 2 mM l -glutamine and 25 µg/ml streptomycin, and 25 U/ml penicillin, 10 ng/ml mouse epidermal growth factor (PeproTech) and 1 µg/ml hydrocortisone (Sigma-Aldrich). Medium for HMEC1-hFcRn was also supplemented with 5 µg/ml blasticidin (InvivoGen) and 100 µg/ml G418 (Sigma-Aldrich) to maintain stable expression of hFcRn. High five cells (Invitrogen) were grown in Express FIVE SEF medium (Invitrogen) supplemented with 18 mM l -glutamine and 1% antibiotic–antimyotic (Invitrogen). All cell lines were negative for mycoplasma contamination (MycoAlertTM PLUS Mycoplasma detection kit, Lonza).
10
Culture of Mouse Spermatogonial Stem Cells
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The mouse SSC line [22 (link)], a kind gift from Professor Fan LQ (Institute of Reproductive & Stem Cell Engineering, Central South University, Changsha, Hunan, China), was cultured in StemPro-34 SFM (Invitrogen, CA, USA), supplemented with 100 μg/mL transferrin, 25 μg/mL insulin, 30 nM sodium selenite, 2 mM L-glutamine, 20 ng/mL mouse epidermal growth factor (PeproTech Ltd., NJ, USA), 20 ng/mL mouse basic fibroblast growth factor (PeproTech Ltd.), and 15 ng/mL recombinant human GDNF (PeproTech Ltd.), on mouse embryonic fibroblasts (MEF) feeders, as described in a previous study [23 (link)]. All cultures were maintained at 34°C in a humidified 5% CO2 incubator. The medium was changed every 2-3 days and the cells were passaged at 7-day intervals with a ratio of 1 : 2-1 : 3.
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