Freshly isolated CD45neg c-kitpos cardiac cells were plated in gelatin-coated dishes in CSC growth medium9 (link), 15 (link) consisting of Dulbecco’s MEM/Ham’s F12 (DMEM/F12; Sigma, St. Louis, MO, USA) medium containing 10% ESQ-FBS (Life Tech, ThermoScientific, Waltham, MA, USA), LIF (10 ng/ml; Miltenyi), FGF (10 ng/ml; Peprotech, Rocky Hill, NJ, USA), EGF (20 ng/ml; Peprotech), insulin–transferrin–selenite (Life Tech), EPO (2.5 U; Sigma, only for rat cells), 1% pen-strep (Life Tech) and 0.1% gentamicin (10 mg/ml liquid, Life Tech). Except where indicated (low oxygen), cells were maintained at 37 °C in ambient O2 (21%) and 5% CO2. Media were replenished every 48 h and cells were passaged at a 1:4 ratio.
Esq fbs
Manufactured by Thermo Fisher Scientific
Sourced in United States, Italy
The ESQ-FBS is a laboratory equipment product designed for sample preparation and analysis. It features a compact and robust design to perform essential functions in a laboratory setting. The core function of the ESQ-FBS is to facilitate sample processing and preparation tasks.
Automatically generated - may contain errors
Lab products found in correlation
Chir99021, by Axon Medchem (3 mentions)
Pd0325901, by Axon Medchem (3 mentions)
Gentamicin, by Thermo Fisher Scientific (2 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Knockout dmem, by Thermo Fisher Scientific (2 mentions)
Fibroblast growth factor (fgf), by Thermo Fisher Scientific (1 mentions)
Pen strep, by Thermo Fisher Scientific (1 mentions)
Magnetic activated cell sorting (macs), by Miltenyi Biotec (1 mentions)
Anti mouse cd45 antibody, by Miltenyi Biotec (1 mentions)
Uo126, by Cell Signaling Technology (1 mentions)
Alpha mem, by Thermo Fisher Scientific (1 mentions)
Ascorbic acid, by Merck Group (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
β glycerophosphate, by Merck Group (1 mentions)
Fungizone, by Thermo Fisher Scientific (1 mentions)
Insulin transferrin selenium, by Thermo Fisher Scientific (1 mentions)
N2 supplement, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
6 protocols using esq fbs
1
Isolation and Culture of Cardiac Progenitor Cells
2
Cardiac Stem Cell Isolation and Differentiation
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CSCs were isolated from adult transgenic and wt hearts by enzymatic methods.35 (link), 40 (link) For CD45−c-Kit+ cell purification, myocyte-depleted small cardiac cells were incubated with microbeads conjugated with anti-mouse CD45 antibody (Miltenyi Biotec S.r.l., Calderara di Reno (BO), Italy) and removed from the preparation by magnetic-activated cell sorting (Miltenyi). From the CD45− fraction, the c-Kit+ (CD45−) cardiac cells were enriched through incubation with a microbeads-conjugated mouse monoclonal antibody against c-Kit (Miltenyi) and separated by magnetic-activated cell sorting.
Freshly isolated CD45−c-Kit+ cardiac cells were cultured on gelatin-coated dishes in CSC growth medium35 (link), 40 (link) before clonogenic, spherogenic and BrdU assays.
Cardiosphere myogenic differentiation was performed as previously described.35 (link)Endothelial differentiation was obtained by culturing the CSC for 3–10 days in MEM Alpha (Life Technologies), 10% ESQ-FBS (Life Technologies), 1 μM dexamethasone, 50 μM/ml ascorbic acid, 10 mM β-glycerophosphate (all from Sigma-Aldrich, Milano, Italy) and 10 ng/ml VEGF (PeproTech EC Ltd., London, UK). LY294002 (10 μM, Calbiochem, San Diego, CA, USA) and UO126 (10 μM, Cell Signalling Technology, Danvers, MA, USA) were added to the culture for AKT and ERK1/2 inhibition.
Freshly isolated CD45−c-Kit+ cardiac cells were cultured on gelatin-coated dishes in CSC growth medium35 (link), 40 (link) before clonogenic, spherogenic and BrdU assays.
Cardiosphere myogenic differentiation was performed as previously described.35 (link)Endothelial differentiation was obtained by culturing the CSC for 3–10 days in MEM Alpha (Life Technologies), 10% ESQ-FBS (Life Technologies), 1 μM dexamethasone, 50 μM/ml ascorbic acid, 10 mM β-glycerophosphate (all from Sigma-Aldrich, Milano, Italy) and 10 ng/ml VEGF (PeproTech EC Ltd., London, UK). LY294002 (10 μM, Calbiochem, San Diego, CA, USA) and UO126 (10 μM, Cell Signalling Technology, Danvers, MA, USA) were added to the culture for AKT and ERK1/2 inhibition.
3
Isolation and Expansion of Cardiac Stem Cells
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Mouse Cardiac stem cells (CSCs) were isolated from adult mouse hearts by enzymatic dissociation using gentleMACS Dissociator (Miltenyi Biotec) as previously described 36, 48 . Cloned CSCs were generated by single cell deposition into wells of 96-well gelatin coated Terasaki plates by serial dilution 36, 48 . Mouse CSCs clones were expanded on gelatin-coated dishes in CSCs growth medium containing of a 1:1 ratio of Neurobasal medium (Gibco, Life Technologies) and DMEM-F12Ham's (Gibco, Life Technologies) implemented with insulin-transferrin-selenium (1%, Life Technologies), epidermal growth factor ( nal medium concentration: 20 ng/ml, Peprotech), basal broblast growth factor ( nal medium concentration: 10 ng/ml, Peprotech) and leukemia inhibitory factor ( nal medium concentration: 10 ng/ml, Miltenyi Biotec) and containing 37 mg of L-glutamine, B27 supplement (2%, Life Technologies) and N2 supplement (1%, Life Technologies) penicillin-streptomycin (1%, Life Technologies), Fungizone (0.1%, Life Technologies) and gentamicin (0.1%, Life Technologies) sterilize through a 0.22-µm pore lter into a sterile container and stored at 4°C. The CSCs growth medium was supplemented with 10% ESQ-FBS (Life Technologies). Cells were maintained at 37°C in ambient O2 (21%) and 5% CO2. Media were replenished every 48 hours and cells were passaged at a 1:4 ratio.
4
Conditional Knockout mESC Generation
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The Rosa26Cre-ERT2/+; Taf8flox/flox mouse embryonic stem cells (mESCs) were generated previously by F. El Saafin21 (link). Briefly, mice carrying the Taf8lox allele were bred to mice carrying the Rosa26Cre-ERT2 allele to produce Rosa26Cre-ERT2/+;Taf8flox/flox E3.5 blastocysts and to isolate Rosa26Cre-ERT2/+;Taf8flox/flox mouse embryonic stem cells (mESCs)21 (link). The Rosa26Cre-ERT2/R;Taf10flox/flox mESCs were generated previously by P. Bardot20 (link). Briefly, the ESCs were derived from Rosa26Cre-ERT2/R;Taf10lox/lox E3.5 blastocysts20 (link). mESCs were cultured in DMEM (4.5 g/l glucose) with 2 mM Glutamax-I, 15% ESQ FBS (Gibco), penicillin, streptomycine, 0.1 mM non-essential amino acids, 0.1% ß-mercaptoethanol, 1500 U/mL LIF and two inhibitors (2i; 3 µM CHIR99021 and 1 µM PD0325901, Axon MedChem) on gelatin-coated plates. To induce deletion of Taf8, mESCs were treated with 0.5 µM 4-OH tamoxifen (Sigma) for 5–6 days, and to induce deletion of Taf10, Rosa26Cre-ERT2/R;Taf10lox/lox mESCs were treated for 4 days with 0.1 µM 4-OH tamoxifen (Sigma). The above-described mESCs have already been described20 (link),21 (link) and were derived according to animal welfare regulations and guidelines of the French Ministry of Agriculture and French Ministry of Higher Education and Research, and the Australian Animal Welfare Committee, respectively.
5
Culturing Human and Mouse Stem Cells
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Human HeLa cells (CCL-2; ATCC) were obtained from the IGBMC cell culture facility and cultured in DMEM (4.5 g/L glucose) supplemented with 10% fetal calf serum (Dutscher, S1810) and 100 U/mL penicillin 100 μg/mL streptomycin (Invitrogen, 15140–130). E14 mouse embryonic stem cells (mESCs, ES Parental cell line E14Tg2a.4, Mutant Mouse Resource and Research Center) were obtained from the IGBMC cell culture facility and cultured on gelatinized plates in feeder-free conditions in KnockOut DMEM (Gibco) supplemented with 20 mM L-glutamine, pen/strep, 100 μM non-essential amino acids, 100 μM β-mercaptoethanol, N-2 supplement, B-27 supplement, 1000 U/mL LIF (Millipore), 15% ESQ FBS (Gibco) and 2i (3 μM CHIR99021, 1 μM PD0325901, Axon MedChem). Cells were grown at 37°C in a humidified, 5% CO2 incubator.
6
Culturing HeLa and mESC cells
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Human HeLa cells (CCL-2; ATCC) were obtained from the IGBMC cell culture facility and cultured in DMEM (4.5 g L–1 glucose) supplemented with 10% fetal calf serum (Dutscher, S1810), 100 U ml penicillin and 100 μg ml–1 streptomycin (Invitrogen, 15140-130). E14 mESCs (ES Parental cell line E14Tg2a.4, Mutant Mouse Resource and Research Center) were obtained from the IGBMC cell culture facility and cultured on gelatinized plates in feeder-free conditions in KnockOut DMEM (Gibco) supplemented with 20 mM l -glutamine, penicillin–streptomycin, 100 µM non-essential amino acids, 100 µM β-mercaptoethanol, N-2 supplement, B-27 supplement, 1000 U ml–1 LIF (Millipore), 15% ESQ FBS (Gibco) and 2i (3 µM CHIR99021, 1 µM PD0325901, Axon MedChem). Cells were grown at 37 °C in a humidified, 5% CO2 incubator.
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