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.
R cm 561
Manufactured by Lonza
The R-CM-561 is a laboratory instrument used for the analysis and processing of samples. It is designed to perform various functions related to sample preparation, measurement, and data processing. The core function of the R-CM-561 is to provide accurate and reliable results for research and analytical applications. Detailed specifications and intended use are not provided to maintain an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Hl 1 cells, by Merck Group (1 mentions)
H9c2 cell, by American Type Culture Collection (1 mentions)
Claycomb medium, by Merck Group (1 mentions)
Mycoalert mycoplasma detection kit, by Lonza (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Aggrewell 400 plate, by STEMCELL (1 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (1 mentions)
Ab692, by Abcam (1 mentions)
Ab25901, by Abcam (1 mentions)
Ab152130, by Abcam (1 mentions)
Ab106393, by Abcam (1 mentions)
Vybrant dio cell labeling solution, by Thermo Fisher Scientific (1 mentions)
Ab24533, by Abcam (1 mentions)
Ary009, by R&D Systems (1 mentions)
Wst 8 assay, by Dojindo Laboratories (1 mentions)
5 protocols using r cm 561
1
Cell Culture Protocols for Cardiac Research
2
Immunological and Cardiomyocyte Dynamics
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RV and lung tissue were prepared for immunohistochemistry, flow cytometry, or Western blotting. Peripheral blood mononuclear cells were evaluated via flow cytometry or used to obtain enriched monocytes tested in vitro for the activation of NLRP3 or cocultured with rat neonatal cardiomyocytes (R-CM-561, Lonza). Cardiomyocytes were assessed for number, size, mitochondrial fragmentation, and membrane potential (see online supplement; Table E1).
3
Cardiac Cell Spheroid Formation
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We prepared spheroids using primary isolated cardiac cells (Lonza R‐CM‐561). Cells were formed into spheroids using Aggrewell 400 plates (Stem Cell Technologies), as per the manufacturer's instructions. In brief, Aggrewell plates were coated with anti‐adherence medium (Lonza) for 30 min at room temperature, before the anti‐adherence medium was removed and the Aggrewells rinsed with Phosphate Buffered Saline (PBS) (Thermofisher Scientific). PBS was replaced with growth media (Lonza Rat Cardiomyocyte Basal Medium (CC‐3275) supplemented with 6.5% V/V Fetal Bovine Serum (Gibco), 6.5% V/V Horse Serum, and 1% V/V Antibiotic Antimycotic (Gibco)) containing cardiac cells, diluted to achieve a final concentration of 50 to 100 cells per microwell, representing small and large spheroids. Spheroids were left to form overnight in an incubator at 37°C for use the next day.
4
Establishment of Cell Culture Models
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Two groups of cells were considered for the experiments: (i) H9c2 in co-culture with NOR-10 (fibroblast) cells as the first model to set up the system, and (ii) neonatal cardiac cells from rats for functionality tests. The H9c2 cell line was obtained from the European Collection of Authenticated Cell Cultures (ECACC) (Lot# 17A028) and cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS), 1% penicillin and streptomycin in 75 cm2 tissue culture flasks at 37 °C, and 5% CO2 in humidified atmosphere. NOR-10 (ECACC 90112701) cells were obtained from the European Collection of Authenticated Cell Cultures. The cells were cultured in DMEM medium supplemented with 10% fetal bovine serum, 1% penicillin and streptomycin in 75 cm2 tissue culture flasks at 37 °C, and 5% CO2 in an incubator. The neonatal rat cardiac cells were purchased from Lonza (R-CM-561) and cultured in RCBM basal medium supplemented with 7.5% horse serum, 7.5% FBS, and 0.1% GA. The culturing was conducted as suggested by the supplier.
5
Investigating IGF-1's Cytoprotective Role
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The capacity to IGF-1–engineered EDCs to withstand cell death was assessed after culture in hypoxic (1% oxygen), low-serum (1% serum) conditions for 48 hours by examining (1) proliferation using the WST-8 assay (Dojindo); (2) expression of early apoptotic markers using flow cytometry (559763; BD Biosciences); (3) expression of Bcl-2, Fos, and Jun prosurvival transcripts using qPCR (Integrated DNA Technologies); (4) expression of 35 apoptosis-related and stress-activated proteins using a membrane-based antibody array (ARY009; R&D Systems); and (5) expression of necroptosis markers (RIP1, ab106393; RIP3, ab152130; caspase-8, ab25901; FADD, ab24533; Abcam) using Western blot densitometry.
The prosurvival effect of IGF-1 overexpression on neighboring myocardium was explored during direct and indirect (Transwell; Corning) coculture with neonatal rat ventricular myocytes (NRVMs; R-CM-561; Lonza). EDCs were distinguished from NRVMs using Vybrant DiO Cell-Labeling Solution (Molecular Probes). NRVMs and EDC cocultures underwent analysis of (1) cell viability using the colorimetric WST-8 assay, (2) apoptosis using flow cytometry for annexin V, and (3) expression of the antiapoptotic protein Bcl-2 (ab692; Abcam) using immmunohistochemistry.
The prosurvival effect of IGF-1 overexpression on neighboring myocardium was explored during direct and indirect (Transwell; Corning) coculture with neonatal rat ventricular myocytes (NRVMs; R-CM-561; Lonza). EDCs were distinguished from NRVMs using Vybrant DiO Cell-Labeling Solution (Molecular Probes). NRVMs and EDC cocultures underwent analysis of (1) cell viability using the colorimetric WST-8 assay, (2) apoptosis using flow cytometry for annexin V, and (3) expression of the antiapoptotic protein Bcl-2 (ab692; Abcam) using immmunohistochemistry.
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