Human cardiac fibroblasts were seeded into 6-well plates and grown to confluence in supplemented Iscove’s basal medium (Biochrom AG, Berlin, Germany) as described above. Before stimulation, cells were serum-starved in a reduced medium, Iscove’s basal medium with 0.5% fetal calf serum (Biochrom AG, Berlin, Germany), 100 U/mL penicillin, and 100 µg/mL streptomycin (Sigma-Aldrich, Taufkirchen, Germany) overnight. Afterwards, cells were stimulated either with 10 ng/mL recombinant human TNF-α (Peprotech, Hamburg, Germany) or 5 ng/mL recombinant human TGF-β (Peprotech, Hamburg, Germany), diluted in serum-reduced Iscove medium. Cells treated equally without the addition of TNF-α or TGF-β and incubated for the same time periods were used as controls. Depending on the experiment, cells were stimulated from 6 to 72 h.
Iscove s basal medium
Manufactured by Harvard Bioscience
Sourced in Germany
Iscove's basal medium is a cell culture medium formulation designed to support the growth and maintenance of a variety of cell types, including hematopoietic and lymphoid cells. It provides a balanced mixture of nutrients, vitamins, and other essential components required for cell proliferation and survival in vitro.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Harvard Bioscience (4 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (3 mentions)
Streptomycin, by Merck Group (2 mentions)
Penicillin, by Merck Group (2 mentions)
Fetal calf serum (fcs), by Harvard Bioscience (2 mentions)
Stable glutamine, by Thermo Fisher Scientific (2 mentions)
Mycospy kit system, by Biontex (2 mentions)
Collagenase 4, by Thermo Fisher Scientific (2 mentions)
β mercaptoethanol, by Merck Group (2 mentions)
Fbs superior, by Harvard Bioscience (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Recombinant human tgf β, by Thermo Fisher Scientific (1 mentions)
Recombinant human tnf α, by Thermo Fisher Scientific (1 mentions)
Penicillin streptamycin, by Thermo Fisher Scientific (1 mentions)
F actin, by Merck Group (1 mentions)
Axiovert m200 microscope, by Zeiss (1 mentions)
Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
R37117, by Thermo Fisher Scientific (1 mentions)
8 protocols using iscove s basal medium
1
Cardiac Fibroblast Stimulation Assay
2
Murine ES Cell Line Differentiation
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Murine ES cell lines described previously [57 (link)] were grown in high glucose DMEM with stable glutamine (GIBCO) containing 10 % FBS Superior (Biochrom), 100 μM non-essential amino acids (GIBCO), 1 % Penicillin/Streptamycin (GIBCO) and 100 μM β-Mercaptoethanol (Sigma) in presence of 1000 U/mL of Leukemia inhibitory factor (LIF, Milllipore). Differentiation of aCaBs was performed in hanging drop culture for two days using 1000 cells as starting material for one EB in Iscove’s basal medium (Biochrom) containing 10 % FBS (Biochrom), 100 μM non-essential amino acids (GIBCO), 1 % Penicillin/Streptamycin (GIBCO) and 450 μM 1-Thioglycerol. For additional 4 days, the cells were differentiated in suspension culture, and at day 6 of differentiation consistently 15 EBs were seeded on one well of a 24-well-plate. Antibiotic selection with 400 μg/mL G418 (Biochrom) was initiated at day 8 post seeding. 4 days thereafter, aCaBs were isolated via treatment with 6000 U/mL Collagenase IV (GIBCO) for 30 min. To obtain single cells for subsequent experiments, the bodies were further dissociated with 100 % Accutase (Affimetrix) for 15 min. To ensure successful generation of aCaBs, potential mycoplasma contamination was routinely controlled twice a week using the PCR based MycoSPY kit system (Biontex).
3
Immunofluorescence Analysis of Cardiac Fibroblasts
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Human cardiac fibroblasts were seeded in chamber slides and grown to confluence with supplemented Iscove’s basal medium (Biochrom AG, Berlin, Germany) as described above. Cells were stimulated either with TNF-α or TGF-β for 6 h and fixed with 4% paraformaldehyde for subsequent fluorescence staining. Antibodies directed against NF-κB p65 ((D14E12) XP® rabbit mAb #8242) or Smad2/3 ((D7G7) XP® rabbit mAb #8685) (Cell Signaling, Frankfurt, Germany) were used. For visualization, Alexa Fluor 594 conjugated goat anti-rabbit IgG (H+L) was applied as a secondary antibody (R37117, Thermo Fisher Scientific, Schwerte, Germany). To visualize the cell shape, Alexa Fluor® 488 Phalloidin (A12379, Thermo Fisher Scientific) was used to stain F-Actin and Hoechst 33,342 (B2261, Sigma-Aldrich, Taufkirchen, Germany) was used to stain nuclei. Documentation of the staining was done with an Axiovert M200 microscope equipped with an ApoTome modul (Zeiss, Jena, Germany).
4
Isolation and Culture of Human Cardiac Fibroblasts
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Patients with dilated cardiomyopathy (EF < 45%) were included in this study. Before obtaining endomyocardial biopsies, coronary artery disease was excluded and ejection fraction was evaluated by echocardiography. The patient’s characteristics are summarized in Supplemental Table S6 . All patients provided their written informed consent for invasive diagnostic procedures. The research protocol was approved by the local institutional review committee of the Charité Berlin, Benjamin-Franklin Campus (no: 225-07, approved 2008).
Human cardiac fibroblasts were obtained by outgrowth from endomyocardial biopsies as described previously [12 (link),19 (link)]. Cells were cultured in Iscove’s basal medium (Biochrom AG, Berlin, Germany) containing 10% human serum, 10% fetal calf serum (Biochrom AG, Berlin, Germany), 100 U/mL penicillin, and 100 µg/mL streptomycin (Sigma-Aldrich, Taufkirchen, Germany). All experiments with those primary cells were carried out between passages 2 and 3 [12 (link),34 (link)].
Human cardiac fibroblasts were obtained by outgrowth from endomyocardial biopsies as described previously [12 (link),19 (link)]. Cells were cultured in Iscove’s basal medium (Biochrom AG, Berlin, Germany) containing 10% human serum, 10% fetal calf serum (Biochrom AG, Berlin, Germany), 100 U/mL penicillin, and 100 µg/mL streptomycin (Sigma-Aldrich, Taufkirchen, Germany). All experiments with those primary cells were carried out between passages 2 and 3 [12 (link),34 (link)].
5
Derivation and Differentiation of Murine ESCs
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ESC lines were derived from the murine line GSES (David et al., 2005 (link)) and grown in high-glucose DMEM with stable glutamine (GIBCO) containing 10% FBS Superior (Biochrom), 100 μM nonessential amino acids (GIBCO), 1% penicillin/streptomycin (GIBCO), and 100 μM β-mercaptoethanol (Sigma) in the presence of 1,000 U/ml of LIF (Milllipore). Differentiation was performed according to standard protocols in Iscove’s basal medium (Biochrom) containing 10% FBS (Biochrom), 100 μM nonessential amino acids (GIBCO), 1% penicillin/streptomycin (GIBCO), and 450 μM 1-thioglycerol (David et al., 2008 (link)). The cells were passaged by trypsin/EDTA (GIBCO) at 70% confluence, which was usually achieved after 2–3 days. Typically, three or four passages after cell-thawing differentiation were induced. Transfections with 15 μg plasmid DNA were performed on 10 cm tissue culture dishes using JetPEI (Peqlab), with subsequent selection via 10 μg/ml blasticidin (Invivogen) or 250 μg/ml hygromycin (Invitrogen). Stable clones were picked manually, further cultivated, and tested by qRT-PCR and differentiation assays (David et al., 2008 (link)). Positively evaluated clones were subjected to antibiotic treatment for cardiomyocyte enrichment as previously described (Klug et al., 1996 (link)).
6
Suspension Culture of Mouse Embryonic Stem Cells
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Mouse ES cells (line CCE) were grown on mitotically inactivated feeder layers of primary mouse embryonic fibroblasts (purchased from Amsbio, Abingdon, UK) in Iscove's basal medium (Biochrom, Berlin, Germany) supplemented with 15% heat-inactivated (56°C, 30 min) foetal calf serum (FCS) (Sigma-Aldrich), 2 mM glutamine, (PAA, Cölbe, Germany), 100 μM 2-mercaptoethanol (Sigma-Aldrich), 1% (v/v) NEA nonessential amino acid stock solution (Biochrom), 1 mM Na+-pyruvate (Biochrom), 0.4% penicillin/streptomycin (Biochrom), and 1000 U/ml leukemia inhibitory factor (LIF) (Merck Millipore, Darmstadt, Germany) in a humidified environment containing 5% CO2 at 37°C, and passaged every 2-3 days. Adherent cells were enzymatically dissociated using 0.05% trypsin-EDTA in phosphate-buffered saline (PBS) (Thermo Fisher Scientific, Waltham, MA, USA) and seeded at a density of 3 · 106 cells/ml in 250 ml siliconized spinner flasks (CellSpin, Integra Biosciences, Fernwald, Germany) containing 125 ml Iscove's medium supplemented as described above, but devoid of LIF. Following 24 h, 125 ml medium was added to give a final volume of 250 ml. The spinner flask medium was stirred at 20 r.p.m. using a stirrer system (Integra Biosciences). The spinning direction was changed every 1440°. 125 ml cell culture medium was exchanged every day.
7
Differentiation and Isolation of aCaBs and iSABs
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Formation of aCaBs and iSABs was achieved as follows: Differentiation was consistently performed in hanging-drop culture for 2 days using 1,000 cells as the starting material for one EB in Iscove’s basal medium (Biochrom) containing 10% FBS (Biochrom), 100 μM nonessential amino acids (GIBCO), 1% penicillin/streptomycin (GIBCO), and 450 μM 1-thioglycerol. For an additional 4 days, the cells were differentiated in suspension culture, and at day 6 of differentiation, consistently 15 EBs were seeded on one well of a 24-well plate. Antibiotic selection with 400 μg/ml G418 (Biochrom) was initiated at day 8 after seeding and 4 days later, iSABs and aCaBs were isolated via treatment with 6,000 U/ml collagenase IV (GIBCO) for 30 min.
To obtain single cells for subsequent experiments, the bodies were further dissociated with 100% Accutase (Affimetrix) for 15 min.
To ensure successful generation of aCaBs and iSABs, potential mycoplasma contamination was routinely controlled twice a week using the PCR-based MycoSPY kit system (Biontex).
For quantification of spontaneously beating foci per EB, the numbers of EBs successfully attached to the well were scored 1 day after plating. Based on this, beating foci per EB were counted daily. Single beating foci were defined either as being clearly spatially separated from each other or as having different beating frequencies.
To obtain single cells for subsequent experiments, the bodies were further dissociated with 100% Accutase (Affimetrix) for 15 min.
To ensure successful generation of aCaBs and iSABs, potential mycoplasma contamination was routinely controlled twice a week using the PCR-based MycoSPY kit system (Biontex).
For quantification of spontaneously beating foci per EB, the numbers of EBs successfully attached to the well were scored 1 day after plating. Based on this, beating foci per EB were counted daily. Single beating foci were defined either as being clearly spatially separated from each other or as having different beating frequencies.
8
Cytokine Assay with HEK293 Cells
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HEK293T and HEK293 cells were cultured in RPMI 1640 (Biochrom, Germany) supplemented with 10% FBS (Biochrom, Germany) (standard medium) at 37°C and 5% CO2. Cells were transfected using ViaFect transfection reagent (Promega, Germany) according to the manufacture protocol. Supernatant was collected 48 h post-transfection and concentrated using an Amicon Ultra-15 Centrifugal Filter Units according to the manufacturer (Merck, UK).
CEC–NFκB–LUC cells (46 (link)) were cultured in Iscove’s basal Medium (Biochrom, Germany) supplemented with 8% FBS (Biochrom, Germany), 2% chicken serum (Thermo Scientific, Germany), 1% penicillin/streptomycin at 40°C, and 5% CO2.
CEC–NFκB–LUC cells (46 (link)) were cultured in Iscove’s basal Medium (Biochrom, Germany) supplemented with 8% FBS (Biochrom, Germany), 2% chicken serum (Thermo Scientific, Germany), 1% penicillin/streptomycin at 40°C, and 5% CO2.
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