The HL-1 cardiomyocytes-derived cell line [19 (link), 20 (link)] was cultured in Claycomb medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin, 100 g/ml streptomycin, and L-glutamine 2 mM (lnvitrogen, Buenos Aires, Argentina) on plastic dishes coated with 12.5 g/ml fibronectin and 0.02% gelatine (Sigma-Aldrich), under a 5% CO2 atmosphere at 37°C [5 (link)]. The cells underwent fasting in culture medium without FBS for 12 h and then incubated with 100 μg/ml aggLDL for 8 h. To compare, HL-1 cardiomyocytes were also treated with insulin 100 nM for 2 h (Sigma-Aldrich) or Claycomb medium vehicle (control) for different times.
Claycomb medium
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom, Italy
Claycomb medium is a cell culture medium designed for the growth and maintenance of cardiomyocytes, which are heart muscle cells. It is a specialized medium that provides the necessary nutrients and conditions for the optimal growth and development of these cells in vitro.
Automatically generated - may contain errors
Lab products found in correlation
Norepinephrine, by Merck Group (44 mentions)
L glutamine, by Merck Group (25 mentions)
Fetal bovine serum (fbs), by Merck Group (24 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (21 mentions)
L glutamine, by Thermo Fisher Scientific (14 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (14 mentions)
Fibronectin, by Merck Group (13 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (11 mentions)
Penicillin, by Thermo Fisher Scientific (10 mentions)
Penicillin streptomycin, by Merck Group (10 mentions)
Gelatin, by Merck Group (9 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (9 mentions)
Streptomycin, by Thermo Fisher Scientific (7 mentions)
Penicillin, by Merck Group (7 mentions)
Hl 1 cells, by Merck Group (7 mentions)
Streptomycin, by Merck Group (5 mentions)
Glutamax, by Thermo Fisher Scientific (5 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (3 mentions)
Hek293t, by American Type Culture Collection (3 mentions)
Opti mem, by Thermo Fisher Scientific (3 mentions)
147 protocols using claycomb medium
1
Cultivating and Treating HL-1 Cardiomyocytes
2
Culturing HL-1 Cardiomyocytes and Lipid Metabolism
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The HL-1 cardiomyocytes-derived cell line19 (link),20 (link) was cultured in Claycomb medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin, 100 g/mL streptomycin, and l -glutamine 2 mM (Invitrogen, Buenos Aires, Argentina) on plastic dishes coated with 12.5 g/mL fibronectin and 0.02% gelatine (Sigma-Aldrich), under a 5% CO2 atmosphere at 37 °C5 (link). The cells underwent fasting in culture medium without FBS for 12 h and then incubated with 100 µg/mL aggLDL for 8 h. To compare, HL-1 cardiomyocytes were also treated with insulin 100 nM for 2 h (Sigma-Aldrich) or Claycomb medium vehicle (control) for different times.
3
Culturing Endothelial and Cardiac Cells
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TeloHAEC cells were purchased from American Type Culture Collection (ATCC; CRL-4052), and HAEC cells were purchased form Lonza (CC-2535). Both cell types were cultured in endothelial cell growth medium -2 (EGM-2) (Lonza, CC-3162) supplemented with antibiotic-antimycotic (Gibco, 15240062). HEK293T cells (ATCC, CRL-3216) were cultured in Dulbecco’s modified Eagle’s medium (Gibco) supplemented with 2 mM l -glutamine and 10% fetal bovine serum (FBS). HL-1 cells were a gift from I. Moskowitz (University of Chicago, USA) and were cultured in Claycomb medium (Millipore Sigma, 51800C) supplemented with 10% FBS, penicillin-streptomycin, 0.1 mM norepinephrine, and 2 mM l -glutamine. For all experiments, cells were either cultured in a normoxic incubator (20% O2 and 5% CO2) or in an incubator inside of a hypoxia glove box (Coy Lab Products, O2 Control InVitro Glove Box; 1% O2 and 5% CO2).
4
Robust GATA-4-mCherry M-HL-1 Cell Line
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Robust generation of a Gata-4-mCherry knock-in m-HL-1 cell line was performed by using CRISPR-Cas9. CMV-Cas9N-2A-GFP (ATUM, Newark, CA, USA) was used as the nickase expression vector. Guide RNA was designed by UNITECH (Chiba, Japan). M-HL-1 cells were maintained in Claycomb medium (MilliporeSigma) with 10% FBS. M-HL-1 cells were transfected with CRISPR-Cas9 and gene-targeting plasmids using FuGENE6 transfection reagent following the manufacturer’s instructions. M-HL-1 clones that had stably integrated the targeting vector were identified by selection with 2 μg/ml puromycin. Drug-resistant clones were manually collected into 96-well plates and expanded for genomic DNA extraction and continued culturing. Targeting efficiency was quantified by PCR screening of genomic DNA from drug-resistant clones. Amplicon identity and gene targeting were confirmed by sequencing (UNITECH, Japan).
5
Cell culture maintenance and thyroid hormone depletion
6
HL-1 Cell Line Catecholamine Overstimulation
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The HL-1 immortalized cell line (SCC065, Merck, St. Louis, MO, United States) was cultured in Claycomb medium (51800C, Merck) according to an optimized protocol provided by Dr. Claycomb’s laboratory (33 (link)). For all experiments, passages 9–12 were used with at least 80% confluency (approximately four days after seeding). For catecholamine overstimulation, cells were exposed to 0.01, 0.1, 0.5, 1, or 2.5 mM ISO (in Claycomb medium with supplements) for 2 or 24 h.
7
Standardizing EV Preparations by Lipid Content
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The purity of the EV fractions was assessed by measuring the total protein and total lipid contents [28 (link)]. The protein content of the EVs was determined using a microBCA Protein Assay kit (ThermoFisher, Waltham, MA, USA), according to the instructions of the manufacturer. The total lipid content was measured using a Lipid Detection Kit (Bioxol, Budapest, Hungary) as previously described [7 (link)]. The serum-free Claycomb medium (Merck, Darmstadt, Germany) of the HL1 cells contains a high concentration of proteins (e.g., bovine albumin) [29 (link)]; consequently, significant amounts of proteins may adsorb onto the EV surface as part of their protein corona [30 (link),31 (link)]. Due to this unknown external protein cargo, we decided to standardize the HL1-derived EV preparations based on their lipid content only. Samples were excluded from further investigations based on an arbitrarily determined total lipid lower threshold of 1 µg.
8
Murine Atrial Cardiomyocyte HL-1 and Rat B-13 Cell Culture
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The murine atrial cardiomyocyte HL-1 cell line (Claycomb et al., 1998 (link)) was routinely cultured in Claycomb medium (Sigma, Poole UK) containing 10% (v/v) fetal calf serum (FCS), 80 units/ml penicillin, 80 μg/ml streptomycin and 0.584 g/l l -glutamine. HL-1 cells were sub-cultured after washing cells with HBSS and incubation with trypsin-EDTA solution (0.05% trypsin in 0.02% EDTA-Na) (Sigma, Poole, UK). An equal volume of soybean trypsin inhibitor was added to inactivate trypsin digestion and the cells were harvested in FCS- and norepinephrine-free Claycomb medium, centrifuged at 500 g for 5 min and cell pellets resuspended and cultured in complete Claycomb medium. Media was replenished daily. Rat B-13 cells were cultured in low glucose (1000 mg/L) Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% (v/v) fetal calf serum (FCS), 80 units/ml penicillin, 80 μg/ml streptomycin and 0.584 g/l l -glutamine. For a review of the cell line, see Probert et al. (2015) . All cell lines were incubated at 37 °C in a humidified atmosphere of 5% CO2 in air. For additional information regarding the MTT reduction as a proxy for cell viability, see supplementary data .
9
Hypoxia Stimulation of HL-1 Mouse Cardiomyocytes
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The HL-1 (non-beating) mouse cardiomyocyte cell line was a kind gift from Professor Enn Seppet (Tartu, Estonia) and was maintained as described previously (Claycomb et al., 1998) .
Cells were grown in supplemented Claycomb medium (Sigma) including HCO3 -, in an atmosphere of 5% CO2 for 24-36 hours until 70% confluent. For hypoxic stimuli, medium was replenished and cells were incubated in a hypoxia chamber (Billups-Rothenberg, Inc.) for 48 hours. Oxygen partial pressure, measured by an O2 sensitive probe (Oxymicro, WPI) was set to 7 mmHg (1% O2) and CO2 was held at 5% CO2 by delivering 95% N2 and 5% CO2 gas mixture.
After 24 hours, the chamber was re-flushed with a fresh mixture of gas. To activate hypoxic pathways using pharmacology, cells were incubated in 21% (normoxia) O2 (145 mm Hg) in the presence of 1 mM 2-oxoglutarate-dependent dioxygenase inhibitor dimethyloxalylglycine (DMOG, Sigma) for 48 hours. Control cells were incubated in 21% O2 for 48 hours. Metabolism will tend to acidify media, but the extent of this was diluted by using large volumes of media in 60 mm petri dishes containing 6 ml Claycomb medium. For NHE activity measurements, HL-1 cells were first grown on coverslips, which were then transferred to 6 well plates containing 2 ml medium.
Cells were grown in supplemented Claycomb medium (Sigma) including HCO3 -, in an atmosphere of 5% CO2 for 24-36 hours until 70% confluent. For hypoxic stimuli, medium was replenished and cells were incubated in a hypoxia chamber (Billups-Rothenberg, Inc.) for 48 hours. Oxygen partial pressure, measured by an O2 sensitive probe (Oxymicro, WPI) was set to 7 mmHg (1% O2) and CO2 was held at 5% CO2 by delivering 95% N2 and 5% CO2 gas mixture.
After 24 hours, the chamber was re-flushed with a fresh mixture of gas. To activate hypoxic pathways using pharmacology, cells were incubated in 21% (normoxia) O2 (145 mm Hg) in the presence of 1 mM 2-oxoglutarate-dependent dioxygenase inhibitor dimethyloxalylglycine (DMOG, Sigma) for 48 hours. Control cells were incubated in 21% O2 for 48 hours. Metabolism will tend to acidify media, but the extent of this was diluted by using large volumes of media in 60 mm petri dishes containing 6 ml Claycomb medium. For NHE activity measurements, HL-1 cells were first grown on coverslips, which were then transferred to 6 well plates containing 2 ml medium.
10
Hypoxia-Reoxygenation Model of HL-1 Cardiomyocytes
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The mouse cardiomyocyte cell line HL-1 (cat. no. SCC065) was purchased from Sigma-Aldrich (Merck KGaA). HL-1 cells were cultured in Claycomb medium (Sigma-Aldrich; Merck KGaA) supplemented with 10% heat-inactivated fetal bovine serum (MP Biomedicals LLC.), 0.1 mM norepinephrine and 1% penicillin-streptomycin (Nacalai Tesque Inc.) in a 5% CO2 humidified incubator at 37°C. To establish a hypoxia/reoxygenation (H/R) model the cells were cultured in a Napco 8000WJ hypoxia (1% O2−5% CO2−94% N2) incubator (Thermo Fisher Scientific, Inc.). After 10 h of hypoxia, the HL-1 cells were plated at 5×105 cells/ml and incubated under normoxic conditions in a CO2 incubator for an additional 4 h. The HL-1 cells passage time was less than 20.
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