Fetal bovine serum (FBS), horse serum (HS), bovine serum albumin (BSA), AC16 human cardiomyocyte cell line (Sigma, St. Louis, MO); αMEM, DMEM/F12, penicillin, streptomycin, gentamycin, amphotericin, l ‐glutamine, HBSS, HEPES, erythrocyte lysing buffer, phosphorylated IGF‐I receptor antibody (Thermo Fisher Scientific/Gibco, Waltham, MA); Total IGF‐I receptor antibody (Novo Biologicals, Littleton, CO); Collagenase type II (Worthington Biochemical Corporation, Lakewood, NJ); IGF‐I, IGF‐II (R&D Systems, Minneapolis, MN); IGFBP‐4 antibodies, phosphorylated Akt antibody, total, and phosphorylated ERK1/2 antibody (Abcam, Cambridge, MA); Total Akt antibody (Cell Signaling, Danvers, MA); Fluorescently labeled secondary antibodies (LI‐COR, Lincoln, NE); IgG2a (Bio X Cell, West Lebanon, NH). Ultrasensitive human PAPPA ELISA (picoPAPP‐A; AL‐101), total human IGFBP‐4 ELISA (AL‐126), and human IGF‐I (AL‐121) ELISA kits as well as an inhibitory PAPPA monoclonal antibody (mAb‐PA 1/41) were generous gifts of Ansh Laboratories (Webster, TX). Recombinant human IGFBP‐4 (wild‐type and protease‐resistant) and recombinant human PAPP‐A (wild‐type and mutated for markedly reduced proteolytic activity) were kindly provided by Professor Claus Oxvig (Aarhus University, Denmark).
Ac16 human cardiomyocyte cell line
Manufactured by Merck Group
Sourced in United States, Germany
The AC16 Human Cardiomyocyte Cell Line is an in vitro cell line derived from human heart tissue. It is a useful tool for the study of cardiac physiology and pathology.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Dmem hams f 12 50 50 mix, by Corning (2 mentions)
Hek293 cell, by American Type Culture Collection (2 mentions)
Penicillin streptomycin, by Corning (2 mentions)
Hcf growth medium, by Cell Applications (2 mentions)
Fluorescently labeled secondary antibody, by LI COR (1 mentions)
Total akt antibody, by Cell Signaling Technology (1 mentions)
Collagenase type 2, by Worthington (1 mentions)
Igg2a, by BioXCell (1 mentions)
Penicillin and streptomycin, by Merck Group (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Pcdna3.1 vector, by Thermo Fisher Scientific (1 mentions)
Anti flag, by Merck Group (1 mentions)
Ripa lysis and extraction buffer, by Thermo Fisher Scientific (1 mentions)
Anti gapdh, by Cell Signaling Technology (1 mentions)
Anti β actin, by Merck Group (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
293t cell, by American Type Culture Collection (1 mentions)
15 protocols using ac16 human cardiomyocyte cell line
1
Cardiomyocyte IGF-I Receptor Signaling
2
Inducing Apoptosis in AC16 Cardiomyocytes
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AC16 Human Cardiomyocyte Cell Line was purchased from Sigma (Saint Louis, MO) and maintained in DMEM/F12 with 4.5 g/L glucose supplemented with 10% (v/v) fetal bovine serum (FBS). Cells were cultured to 80% confluence in a 100 mm dish at 37°C with 5% CO2 before passage and seeding for experiments.
For induction of cell death and apoptosis, AC16 cells were exposed to different concentrations of DOX for various times as indicated, following co-treatment with Wnt10b. The dose of Wnt10b was chosen based on published literature. The 200 nM of 48 hours of DOX-treatment was chosen based on our experiment, as shown inFig 1A .
For induction of cell death and apoptosis, AC16 cells were exposed to different concentrations of DOX for various times as indicated, following co-treatment with Wnt10b. The dose of Wnt10b was chosen based on published literature. The 200 nM of 48 hours of DOX-treatment was chosen based on our experiment, as shown in
3
Myocardial Infarction Plasma Profiling
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Prior to therapy, blood (3ml) was extracted from both healthy controls and MI patients. To prepare plasma samples, blood was mixed with citric acid (1:10), followed by centrifuging the mixture for 15 min at 1200 g to collect the supernatant (plasma). Plasma samples were kept in liquid nitrogen storage prior to the subsequent assays.
Cardiomyocytes and transient transfections AC16 human cardiomyocyte cell line (Sigma-Aldrich) was used in this study. AC16 cells were cultivated in DMEM medium supplemented with 12% FBS and 1% penicillin and streptomycin (Sigma-Aldrich). In an incubator, AC16 cells were cultivated at 37°C, and 95% humidity, a 5% CO 2 .
MFACR and miR-125b were overexpressed in AC16 cells by transfecting AC16 cells with MFACR expression vector or mimic of miR-125b through lipofectamine 2000 (Invitrogen)-mediated transient transfections. MFACR expression vector was constructed with pcDNA 3.1 vector (Invitrogen) as backbone, and mimic of miR-125b and negative control (NC) miRNA were purchased from Sigma-Aldrich. NC experiments (NC miRNA-or empty vector-transfected cells) and C experiments (control cells with no transfections) were included in each experiment. After transfections, AC16 cells were cultivated in fresh medium for further 48h prior to the subsequent experiments.
Cardiomyocytes and transient transfections AC16 human cardiomyocyte cell line (Sigma-Aldrich) was used in this study. AC16 cells were cultivated in DMEM medium supplemented with 12% FBS and 1% penicillin and streptomycin (Sigma-Aldrich). In an incubator, AC16 cells were cultivated at 37°C, and 95% humidity, a 5% CO 2 .
MFACR and miR-125b were overexpressed in AC16 cells by transfecting AC16 cells with MFACR expression vector or mimic of miR-125b through lipofectamine 2000 (Invitrogen)-mediated transient transfections. MFACR expression vector was constructed with pcDNA 3.1 vector (Invitrogen) as backbone, and mimic of miR-125b and negative control (NC) miRNA were purchased from Sigma-Aldrich. NC experiments (NC miRNA-or empty vector-transfected cells) and C experiments (control cells with no transfections) were included in each experiment. After transfections, AC16 cells were cultivated in fresh medium for further 48h prior to the subsequent experiments.
4
Immunoblotting of Cardiac Cell Lines
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For immunoblotting, the cells were lysed in RIPA Lysis and Extraction Buffer (Thermo Fisher Scientific, 89900), quantified by the Lowry method and then 10 µg of proteins were loaded on a 4–20% precast gel. After electrophoretic separation, proteins were transferred onto nitrocellulose membranes that were incubated overnight with primary antibodies including Anti-β-Actin (1:5000, Sigma-Aldrich, A1978), ANTI-Flag® (F7425, Merck), ANTI-GAPDH (1:5000, 2118L, Cell signaling, Denver, MA, USA) and ANTI-Csub (1:1000, ab181243, Abcam). The revelation was assessed by specific HRP-labelled secondary antibodies, followed by detection by chemiluminescence using ChemiDoc™ Touch Gel Imaging System. The Western blots shown in figures are representative of at least three different independent experiments.
Cardiac cell lines lysates: Human Aortic Endothelial Cells (HAEC) were provided by Thermo-Fisher (C0065C) and cultured according to the manufacturer’s instructions; AC16 Human Cardiomyocyte Cell Line was provided by Merck (SCC109) and cultured according to the manifacturer’s instructions; Human Cardiac Myocytes (HCM) were provided by Promocell (FB60C12810 Heidelberg, Germany) and cultured according to the manufacturer’s instructions.
Cardiac cell lines lysates: Human Aortic Endothelial Cells (HAEC) were provided by Thermo-Fisher (C0065C) and cultured according to the manufacturer’s instructions; AC16 Human Cardiomyocyte Cell Line was provided by Merck (SCC109) and cultured according to the manifacturer’s instructions; Human Cardiac Myocytes (HCM) were provided by Promocell (FB60C12810 Heidelberg, Germany) and cultured according to the manufacturer’s instructions.
5
Hypoxic Cardiomyocyte Culture and Analysis
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AC16 Human Cardiomyocyte Cell Line (SCC109, Merck, St. Louis, MO, USA) was cultured in DMEM/F12 medium (Gibco™, Thermo Fisher Scientific, Horsham, UK), supplemented with 2 mM L-Glutamine (Gibco™, Thermo Fisher Scientific), 12.5% fetal bovine serum (FBS, LINUS), 50 U/mL penicillin and 50 µg/mL streptomycin (Gibco™, Thermo Fisher Scientific, Horsham, UK), at 37 °C in a 5% CO2 incubator. The culture medium was replaced every two to three days until 90% cell confluence was reached and was used in the following experimental assays.
In total, 8 × 104 human cardiomyocytes per well were seeded in 6-well plates. The cells were cultured at 37 °C in hypoxic conditions (5% CO2 and 2% O2), simulating the myocardial hypoxia of ICM, or normoxic conditions as control. At 48 h, the cells were harvested and the expression of the target molecules was analyzed.
In total, 8 × 104 human cardiomyocytes per well were seeded in 6-well plates. The cells were cultured at 37 °C in hypoxic conditions (5% CO2 and 2% O2), simulating the myocardial hypoxia of ICM, or normoxic conditions as control. At 48 h, the cells were harvested and the expression of the target molecules was analyzed.
6
AC16 Cells under Hypoxic Conditions
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The AC16 human cardiomyocyte cell line was purchased from EMD Millipore and the human renal epithelial derived 293T cells were purchased from American Type Culture Collection (Bethesda, MD, U.S.A.). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 12% FBS, and 1% antibiotics (streptomycin and penicillin). AC16 cells were incubated at 37°C in a humid atmosphere with 5% CO2 and 95% air. Cells were placed in an Invivo200 cultivator (Ruskin Technology Ltd, U.K.) containing a gaseous mixture of 94% N2, 5% CO2, and 1% O2 at 37°C for durations of 8, 16, 24, 48, and 72 h, respectively. Cells in normoxia group were incubated under the same conditions.
7
Cardiomyocyte Culture with Lactate and Glucose
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AC16 Human Cardiomyocyte Cell Line, sodium l -lactate (L7022) and d -glucose (G7021) were purchased from Merck (Darmstadt, Germany). Dulbecco's Modified Eagle's Medium (DMEM) low glucose (ECM007L) was purchased from Euroclone (Milan, Italy), while DMEM without d -glucose and sodium pyruvate were purchased from Gibco (11,966–025, Thermo Fisher Scientific, Waltham, MA, USA).
8
Culturing AC16 Cardiomyocytes under Normoxia and Hypoxia
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The AC16 human cardiomyocyte cell line, purchased from EMD Millipore (Billerica, MA, USA), was cultured following standard manufacturer protocol. The cells (1×106 cells/well) were seeded in 10% FBS (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and DMEM/F-12 medium (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and allowed to grow overnight. The AC16 CMs were then incubated in either a normoxic condition (37°C, 20% O2 and 5% CO2) or hypoxic condition (37°C, 1% O2 and 5% CO2) for 24 h. The hypoxic condition was maintained in a physiological oxygen workstation (InvivO2 400; Baker Ruskinn, Sanford, ME, USA).
9
Culturing Human Cardiomyocytes from Tissue
10
C3 Expression and Cardiomyocyte Viability
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The AC16 human cardiomyocyte cell line (Millipore, MA, USA) was grown in DMEM:F12 with 10% heat-inactivated FBS, 1% penicillin, and 1% L-Glutamine. The 293 T cell line was grown in DMEM with 10% heat-inactivated FBS and 1% penicillin. Both cell lines were maintained at 37 °C in 5% CO2. Rabbit anti-human C3c (F0201) polyclonal Ab was obtained from Agilent Dako (Santa Clara, CA, USA). Goat anti-human C3 (A213) polyclonal Ab was obtained from Complement Technologies (Tyler, TX, USA). Rabbit anti-cytochrome c polyclonal Ab was obtained from Cell Signaling (Danvers, MA, USA). Rabbit anti-caspase 3 polyclonal Ab was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The full length human complement C3 expression ORF clone, whose C3 gene cDNA ORF clone sequences were retrieved from the NCBI Reference Sequence Database with the vector pcDNA 3.1+/C-(K)DYK, was obtained from GenScript (Piscataway, NJ, USA) (Supplementary Figure S4 ).
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