HNDFs (Gibco BRL, USA; isolated from the skin of newborn male) were plated at a density of 2.5 x 103 cells/cm2 and cultured in Medium 106 (Gibco BRL) supplemented with low serum growth supplement (Gibco BRL) at 37 °C in humid air with 5 %(v/v) CO2. The medium was replaced every other day until the culture is approximately 80 % confluent, and after that, exchanged every day. HL-1 cells (EMD Millipore, USA), murine cardiomyocyte cell line, were cultured in Claycomb medium (Sigma Aldrich) containing 10 %(v/v) fetal bovine serum (FBS; Gibco BRL), 0.1 mM norepinephrine (Sigma Aldrich), 2 mM L-glutamine (Gibco BRL), and 100 units/ml penicillin, and 100 μg/ml streptomycin (Gibco BRL). HL-1 cells were maintained at a high cell density to prevent dedifferentiate, so they were passaged at 1:3 split ratio only at 100% confluency. The culture plates for HL-1 cells were coated with 0.02 %(w/v) gelatin (Sigma Aldrich) and 5 μg/mL fibronectin (Sigma Aldrich).
Hl 1 cells
Manufactured by Merck Group
Sourced in United States, Italy
HL-1 cells are a cardiac muscle cell line derived from the AT-1 mouse atrial cardiomyocyte tumor. They are capable of spontaneous contractions and express cardiac-specific markers, making them a useful tool for the study of cardiac physiology and pathology in a cell culture model.
Automatically generated - may contain errors
Lab products found in correlation
Claycomb medium, by Merck Group (8 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (6 mentions)
Penicillin, by Merck Group (2 mentions)
Streptomycin, by Merck Group (2 mentions)
H9c2 cell, by American Type Culture Collection (2 mentions)
Penicillin streptomycin, by Lonza (2 mentions)
Fetal bovine serum (fbs), by Euroclone (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Low serum growth supplement, by Thermo Fisher Scientific (2 mentions)
Fibronectin, by Merck Group (2 mentions)
Hndfs, by Thermo Fisher Scientific (2 mentions)
Medium 106, by Thermo Fisher Scientific (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Gelatin, by Merck Group (2 mentions)
Norepinephrine, by Merck Group (2 mentions)
Dmem f12, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Merck Group (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
23 protocols using hl 1 cells
1
Culturing HNDF and HL-1 Cardiomyocytes
2
HL-1 Cardiomyocyte Cell Culture Protocol
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HL-1 cells (kindly provided by Dr. William Claycomb, Louisiana State University Health Science Center, New Orleans, LA, USA) originate from atrial tumor cells of transgenic mice and exhibit cardiomyocyte-specific ion channel expression.20 (link),21 (link) KCa2 channel expression in HL-1 cells has been shown previously.9 (link),16 (link) Cells were cultured in a supplemented Claycomb medium (Sigma-Aldrich, Steinheim, Germany) containing 0.1 mM norepinephrine.20 (link) A total of 4–5×106 HL-1 cells per well were seeded on gelatin-/fibronectin-coated 6-well dishes and subjected to experimental procedures, unless stated otherwise. For ß-adrenergic stimulation 50 µM isoproterenol was added to the culture medium for 24 h. Similar doses of isoprenaline have been employed previously in experimental studies.22–24 (link) For HL-1 cell authentication, Short Tandem Repeat (STR) profiling of HL-1 cells used in this study (obtained directly from Dr. Claycomb) and of commercially available HL-1 cells (Sigma-Aldrich, Steinheim, Germany) for comparison was performed by American Type Culture Collection (ATCC) cell-line authentication service (Wesel, Germany). This approach confirmed a high degree of similarity between cell lines (Supplementary Table 2
3
Cultivation of Murine Cardiomyocyte HL-1 Cells
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HL-1 cells (a murine cardiomyocyte cell line, Sigma-Aldrich, Vienna, Austria) were cultured in fibronectin (0.5% [w/v])/gelatin (0.02% [w/v]) coated flasks and maintained in Claycomb medium (Sigma-Aldrich) (Claycomb et al., 1998 (link)) containing 10% (v/v) fetal bovine serum (FBS, Thermo Fisher Scientific, IL, United States), 0.1 mM norepinephrine, 2 mM L-glutamine, 100 IU/ml penicillin and 100 μg/ml streptomycin (Sigma-Aldrich), and kept at 37°C under 5% CO2, as previously described (Scheruebel et al., 2014 (link)).
4
Evaluating HMGB-1 Effects on Murine Cardiac Cells
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For in vitro experiments, the murine cardiac muscle cell line (HL-1 cells) (Sigma Aldrich, St. Louis, MO, USA) was used. Murine HL-1 cells were cultured in HL-1 expansion medium at 37 °C in an atmosphere of 5% CO2. Following this, HL-1 cells were incubated with HMGB-1 (R&D Systems) for 6 h. Cell viability was detected by using a Cell Titer-Glo® Luminescent Cell Viability Assay (Promega, Madison, WI, USA) in the presence of different concentrations of HMGB-1 (1 µg/mL, 100 ng/mL, 10 ng/mL). Moreover, we analysed the metabolic activity of the cells by MTT assay (Invitrogen, Waltham, MA, USA) in the presence of HMGB-1 dose-dependently (after 24 h of incubation). For all experiments n = 6.
5
Cell Culture Protocols for Cardiac Research
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H9c2 cells (ATCC, CRL-1446) were cultured in DMEM supplemented with 10% FBS, penicillin (100 units/mL) and streptomycin (100 μg/mL). HL-1 cells (Sigma, SCC065) were cultured in Claycomb medium (Sigma, 51800 C) supplemented with 10% FBS, penicillin (100 units/mL), streptomycin (100 μg/mL), norepinephrine (0.1 mM), and L-glutamine (2 mM) in fibronectin–gelatin-coated flasks. HL-1 cells were tested negative when examined by MycoAlert mycoplasma detection kit (Lonza). Neonatal rat ventricular cardiomyocytes (P1-2) (Lonza, R-CM-561) were cultured on nitrocellulose-coated plates with the provided medium (Lonza, CC-4515). Cells were cultured at 37 °C in 5% CO2 humidified atmosphere.
6
Cardiomyocyte Stress Response to LPS
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For the in vitro experiments, the murine cardiac muscle cell line (HL-1 cells) [25 (link)] (Sigma Aldrich, St. Louis, MO, USA) and human cardiomyocytes (iPS) (Cellular Dynamics, Madison, WI, USA) were used. Murine HL-1 cells were cultured in an HL-1 expansion medium at 37°C in an atmosphere of 5% CO2. Human cardiomyocytes (iPS) were cultured for 10 days in a maintenance medium (Cellular Dynamics, Madison, WI, USA) at 37°C in an atmosphere of 7% CO2.
Next, HL-1 cells were treated with 20 μg/ml LPS and the human cardiomyocytes with 10 μg/ml LPS for 6 h at 37°C. For the determination of troponin I elevation of cells in the presence of LPS with either ATP or nigericin, the cells were treated for 5 h with LPS with the abovementioned concentrations and for one further hour either with 1 mM ATP (Sigma Aldrich, St. Louis, MO, USA) or with 10 μM nigericin (Sigma Aldrich, St. Louis, MO, USA). The control groups were incubated in cell culture media without any supplements.
Next, HL-1 cells were treated with 20 μg/ml LPS and the human cardiomyocytes with 10 μg/ml LPS for 6 h at 37°C. For the determination of troponin I elevation of cells in the presence of LPS with either ATP or nigericin, the cells were treated for 5 h with LPS with the abovementioned concentrations and for one further hour either with 1 mM ATP (Sigma Aldrich, St. Louis, MO, USA) or with 10 μM nigericin (Sigma Aldrich, St. Louis, MO, USA). The control groups were incubated in cell culture media without any supplements.
7
Tachypacing-Induced Cardiac Hypertrophy in HL-1 Cells
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HL-1 cells were purchased from Sigma-Aldrich (SCC065). HL-1 cells were cultured with Calycomb Medium (Sigma, United States) supplemented with 10% fetal bovine serum (FBS), 1% Penicillin/Streptomycin, 2 mM L-Glutamine, and 0.1 mM Norepinephrine. The cells were cultured in flasks coated with 5 μg/ml fibronectin and 0.02% gelatin at 37°C in a 5% CO2 atmosphere. Then the cells were cultured in six-well dishes to induce tachypacing using a cell pacing system (Ionoptix, USA). The cells were subjected to rapid field stimulation for 24 h at 7 Hz (20 V, 5 ms). In a few experiments, HL-1 cells were pre-treated with 20 nM Fgf21 for 24 h before rapid field stimulation.
8
HL-1 Cell Culture Protocol
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HL-1 cells (Sigma-Aldrich, Milan, Italy) were maintained in Claycomb medium completed with 10% fetal bovine serum (Euroclone, Milan, Italy), 2 mM l-glutamine, 0,1 mM norepirephrine, and 100 μg/mL penicillin/streptomycin (Lonza, Basel, Switzerland). The cells were maintained at 37 °C in a humidified atmosphere of 5% of CO2 in air and subculture until they reached 80% confluence (Video S1 ).
9
Culturing HL-1 Cells and Isolating hGMSCs
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HL-1 cells (Sigma-Aldrich, Milan, Italy) were maintained in Claycomb medium completed with 10% fetal bovine serum (FBS, Euroclone, Milan, Italy), 2 mML-glutamine, 0.1 mM norepirephrine, and 100 μg/mL penicillin/streptomycin (Lonza, Basel, Switzerland). The cells were maintained at 37 °C in a humidified atmosphere of 5% of CO2 in air and subcultured until they reached 80% confluence (Marconi et al., 2022 (link)). Human gingival mesenchymal stem cells (hGMSCs) were isolated from gingival tissues of three patients, in good general health conditions and without oral disease, who underwent surgical procedure. The gingival tissues were placed in a culture dish, fragmented, washed several times in phosphate-buffered saline solution (PBS, Lonza, Basel, Switzerland) with 5% of gentamicin (Lonza) and transferred in the incubator at 37°C in a humidified atmosphere of 5% CO2 in air with mesenchymal stem cell growth medium-chemically defined (MSCGM-CD, Lonza). The medium was replaced every 2 days for approximately 2 weeks before cells reached 80% confluence and were passaged in culture (Marconi et al., 2021c (link)).
10
Transfection of HEK293 and HL1 Cells
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HEK293 cells were obtained from ATCC, and HL1 cells were purchased from Sigma-Aldrich. siRNA for SNRK, UCP3, PPARα and Trib3 were purchased from Dharmacon. Plasmids used in cell culture transfection experiments using Lipofectamine 2000 or Lipofectamine LTX were SNRK-pEGFP-N3, SNRK-T173A-pEGFP-N3, UCP3-pEGFP-N3 and Trib3-pCDNA3.
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