Cc 2583

Manufactured by Lonza

Sourced in United States

The CC-2583 is a lab equipment product manufactured by Lonza. It is designed for general laboratory use, but a detailed technical description is not available at this time.

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Lab products found in correlation

14 protocols using cc 2583

FBS-Induced Signaling in Smooth Muscle Cells

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Human coronary artery smooth muscle cells (CASMC: CC-2583) and human umbilical artery smooth muscle cells (UASMC: CC-2579), purchased from Lonza (Basel, Switzerland), were cultured in smooth muscle growth medium (Lonza SmGM-2: CC-3182) containing 5% fetal bovine serum (FBS), growth factors (hEGF, insulin and hFGF-β), gentamicin and amphotericin-B at 37 °C in a humidified incubator with 5% CO₂. These cells maintain their morphological and phenotypic characteristics for up to 10 passages and were, therefore, used for experiments within 10 passages. Cells were plated at a density of 2 X 10⁵ cells/ml into 4-well plates (Nunc, Thermo Scientific, Waltham, MA) with smooth muscle growth medium containing 5% FBS. Prior to treatment, cells were grown to 70% confluence and starved in FBS-free medium overnight. Cells were then stimulated with 5% FBS for times indicated in the figures (typically 0, 15 s, 30 s, 1 min, 2 min, 5 min, 30 min, 1 h and 2 h) to observe their various responses to FBS. The treated cells were lysed immediately in Laemmli sample buffer and heated at 90 °C for 5 min. To investigate the effects of kinase inhibitors, cells were starved in FBS-free medium for 8 h to induce cell quiescence and then incubated with various inhibitors for ≥ 45 min prior to stimulation with 5% FBS for 2 min and then lysed in Laemmli sample buffer.

Deng J.T., Bhaidani S., Sutherland C., MacDonald J.A, & Walsh M.P. (2019). Rho-associated kinase and zipper-interacting protein kinase, but not myosin light chain kinase, are involved in the regulation of myosin phosphorylation in serum-stimulated human arterial smooth muscle cells. PLoS ONE, 14(12), e0226406.

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Characterization of CagA-Induced Inflammatory Response in CASMCs

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Human coronary artery smooth muscle cells (CASMCs) from 3 donors were commercially obtained (CC-2583; Lonza, Basel, Switzerland). Cell culture reagents, i.e., Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), phosphate-buffered saline (PBS), Penicillin/Streptomycin, Amphotericin B, l-glutamine, HEPES, and trypsin were obtained from ThermoFisher Scientific, Waltham, MA, USA. Recombinant CagA protein containing amino acids 918 to 1147 and an N-terminal His tag was purchased from Abcam, Cambridge, UK. Reagents for the isolation of total RNA were purchased from Qiagen, Hilden, Germany. qPCR reagents were purchased from ThermoFisher Scientific, Waltham, MA, USA. ELISA kits for human IL-1β (DY201) and IL-6 (DY206-05) were purchased from R&D Systems, Minneapolis, MN, USA. IRDye 800CW BoneTag Optical Probe was purchased from LI-COR Biosciences, Lincoln, NE, USA.

Sundqvist M.O., Wärme J., Hofmann R., Pawelzik S.C, & Bäck M. (2023). Helicobacter Pylori Virulence Factor Cytotoxin-Associated Gene A (CagA) Induces Vascular Calcification in Coronary Artery Smooth Muscle Cells. International Journal of Molecular Sciences, 24(6), 5392.

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Osteoblast and C-VSMC Differentiation

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Human bone marrow-derived Mesenchymal Stem Cells (MSCs; PT-2501, Lonza, Walkersville, MD, USA) and Vascular Smooth Muscle Cells (VSMCs; coronary artery smooth muscle cells, CC-2583, Lonza) were cultured as described previously [27 (link)]. Briefly, MSCs and VSMCs were expanded in Mesenchymal Stem Cell Basal Medium (MSCBM, PT-3238, Lonza) supplemented with Mesenchymal Stem Cell Medium SingleQuot Kit (MSCGM, PT-4105, Lonza) and Smooth muscle cell Basal Medium (SmBM, CC-3181, Lonza) supplemented with Smooth muscle Medium-2, SingleQuot Kit (SmGM-2, CC-4149, Lonza) respectively. For induction of MSCs differentiation into osteoblasts (referred also as MSC/osteoblasts) and VSMC development into C-VSMCs (VSMC/C-VSMC), cells were cultured in DMEM medium (GIBCO, Paisley, UK) containing 10% FCS, penicillin/streptomycin, 1.8 mM CaCl₂ (Sigma, St. Louis, MO, USA) and 20 mM HEPES (Sigma), pH 7.5. Additionally, this medium was freshly supplemented with 0.1 mM ascorbic acid (Sigma), 10 mM ß-glycerophosphate (Sigma) and 100 nM dexamethasone (DEX, Sigma). In the present study 2 independent MSC and VSMC donors were used, one for the gene expression array and the other for validation purposes. All analyses were performed on samples collected at the beginning of cell culture (day 0, before induction of differentiation) and during week 1, 2 and 3 of culture.

Alves R.D., Eijken M., van de Peppel J, & van Leeuwen J.P. (2014). Calcifying vascular smooth muscle cells and osteoblasts: independent cell types exhibiting extracellular matrix and biomineralization-related mimicries. BMC Genomics, 15(1), 965.

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Cell Culture Conditions for Macrophages, Endothelial, and Vascular Smooth Muscle Cells

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Mouse macrophages (RAW264.7, ATCC TIB-71) and mouse yolk sac endothelial cells (C166, ATCC CRL-2581) were grown in Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS), while human monocyte cell line (THP-1, ATCC TIB-202) were grown in RPMI-1640 medium containing 10% FBS and 0.05 mM 2-mercaptoethanol. Primary vascular SMCs were harvested from the aortae of C57Bl/6 mice and propagated in DMEM with 10% FBS.^{38 (link)} Human coronary artery SMCs (HCASMCs, Lonza CC-2583) and human aortic endothelial cells (HAECs, Lonza CC-2535) were cultured and maintained according to the manufacturer’s (Lonza) instructions. All cells were cultured in a humidified 5% CO₂ incubator at 37°C. The cell lines were authenticated by the supplier. None of the cell lines were tested for mycoplasma contamination.

Flores A.M., Hosseini-Nassab N., Jarr K.U., Ye J., Zhu X., Wirka R., Koh A.L., Tsantilas P., Wang Y., Nanda V., Kojima Y., Zeng Y., Lotfi M., Sinclair R., Weissman I.L., Ingelsson E., Smith B.R, & Leeper N.J. (2020). Pro-efferocytic nanoparticles are specifically taken up by lesional macrophages and prevent atherosclerosis. Nature nanotechnology, 15(2), 154-161.

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Modulating Quiescent CASMCS with Inhibitors

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Human CASMCS (purchased from Lonza Cat No. CC-2583, Lot No. 0000317155) were grown in DMEM (5mM glucose, 10% FBS and 1% antibiotics at 37°C in 5% CO₂) and were seeded in 60 mm dishes. Cells were grown to confluence then rendered quiescent by serum deprivation for 48 hours. Inhibitors to TGFBR1 (SB431542 10μM) and EGFR (AG1478 5μM) were pre-incubated for 30 mins prior to treatment with thrombin (10 units/ml) for 6 hours. RNA extraction and library preparation followed the manufacturer’s instructions.

Kamato D., Bhaskarala V.V., Mantri N., Oh T.G., Ling D., Janke R., Zheng W., Little P.J, & Osman N. (2017). RNA sequencing to determine the contribution of kinase receptor transactivation to G protein coupled receptor signalling in vascular smooth muscle cells. PLoS ONE, 12(7), e0180842.

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Cell Culture Conditions for Macrophages, Endothelial, and Vascular Smooth Muscle Cells

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Investigating Vascular Cell Types in Coronary Artery Disease

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Human coronary artery VSMCs (Lonza CC-2583; culture media CC-31182) were used at passage five or earlier. Endothelial cell experiments were conducted with immortalized human aortic endothelial cells (ATCC CRL-4052; culture media Lifeline Cell Technology LL-0003). Monocyte experiments were conducted with THP-1 monocyte cells (ATCC TIB-202; culture media RPMI ATCC 30-2001, 10% FBS Sigma 12306C-500ML). Genome editing was performed as previously described (Supplementary Note)^{72 (link)}.

Aragam K.G., Jiang T., Goel A., Kanoni S., Wolford B.N., Atri D.S., Weeks E.M., Wang M., Hindy G., Zhou W., Grace C., Roselli C., Marston N.A., Kamanu F.K., Surakka I., Venegas L.M., Sherliker P., Koyama S., Ishigaki K., Åsvold B.O., Brown M.R., Brumpton B., de Vries P.S., Giannakopoulou O., Giardoglou P., Gudbjartsson D.F., Güldener U., Haider S.M., Helgadottir A., Ibrahim M., Kastrati A., Kessler T., Kyriakou T., Konopka T., Li L., Ma L., Meitinger T., Mucha S., Munz M., Murgia F., Nielsen J.B., Nöthen M.M., Pang S., Reinberger T., Schnitzler G., Smedley D., Thorleifsson G., von Scheidt M., Ulirsch J.C., Arnar D.O., Burtt N.P., Costanzo M.C., Flannick J., Ito K., Jang D.K., Kamatani Y., Khera A.V., Komuro I., Kullo I.J., Lotta L.A., Nelson C.P., Roberts R., Thorgeirsson G., Thorsteinsdottir U., Webb T.R., Baras A., Björkegren J.L., Boerwinkle E., Dedoussis G., Holm H., Hveem K., Melander O., Morrison A.C., Orho-Melander M., Rallidis L.S., Ruusalepp A., Sabatine M.S., Stefansson K., Zalloua P., Ellinor P.T., Farrall M., Danesh J., Ruff C.T., Finucane H.K., Hopewell J.C., Clarke R., Gupta R.M., Erdmann J., Samani N.J., Schunkert H., Watkins H., Willer C.J., Deloukas P., Kathiresan S, & Butterworth A.S. (2022). Discovery and systematic characterization of risk variants and genes for coronary artery disease in over a million participants. Nature Genetics, 54(12), 1803-1815.

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Isolation and Culture of Primary SMCs

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Human primary coronary artery SMCs were obtained from Lonza (catalog number CC-2583) and cultured in smooth muscle basal medium (Lonza; catalog number CC-3181) containing recombinant human FGF basic (5 ng/mL), insulin (5 μg/mL), EGF (5 ng/mL), and FBS (5%). Primary murine SMCs were isolated from the thoracic aorta by enzymatic digestion technique as described previously (47 (link)). Briefly, the thoracic aorta was dissected and cleaned of adventitial tissue and incubated with 1.0 mg/mL collagenase in DMEM at 37°C for 90 minutes. The cell suspension was passed through a 70 μm restrainer, washed, and cultured in DMEM with 10% FBS. Cells were maintained in a 95% air and 5% CO₂ humidified incubator at 37°C for 7 days, trypsinized with 0.25% trypsin-EDTA, and subcultured. All SMCs used for experiments were between the third and fifth passages.

Jain M., Dev R., Doddapattar P., Kon S., Dhanesha N, & Chauhan A.K. (2021). Integrin α9 regulates smooth muscle cell phenotype switching and vascular remodeling. JCI Insight, 6(10), e147134.

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Isolation and Characterization of Human Coronary Cells

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Human primary coronary artery endothelial cells (CAEC) and CAVSMCs from healthy individuals were purchased from Lonza (CC-2545 and CC-2583). Human primary coronary artery vascular smooth muscle cells from T2DM patients with CVD were isolated as previously reported by Dr. Pereira (Dinardo et al., 2015 ; Dinardo et al., 2014 (link); Vaquero et al., 2012 (link)).

Toyohara T., Roudnicky F., Florido M.H., Nakano T., Yu H., Katsuki S., Lee M.J., Torsten M., Friesen M., Davidow L.S., Ptaszek L., Abe T., Rubin L.L., Pereira A.C., Aikawa M, & Cowan C.A. (2020). Patient hiPSCs identify vascular smooth muscle arylacetamide deacetylase as protective against atherosclerosis. Cell stem cell, 27(1), 147-157.e7.

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Adenoviral Regulation of CYR61 in hCSMCs

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Human coronary artery smooth muscle cells (hCSMCs) were purchased from Lonza (CC-2583, Basel, Switzerland). Six- to eight-passage hCSMCs were cultured in SMC growth medium consisting of basal media, insulin, human recombinant epidermal growth factor, human recombinant fibroblast growth factor, gentamicin, amphotericin, and 5% fetal bovine serum at 37°C in an atmosphere of 95% air and 5% CO₂ based on manufacturer's recommendations. Recombinant adenoviral vectors, expressing CYR61 cDNA (Ad-CYR61) and antisense CYR61 cDNA (Ad-AS-CYR61) fragments, were used for the overexpression or suppression of CYR61 gene in cultured hCSMCs. As a control, an adenoviral vector expressing green fluorescence proteins only (Ad-GFP) was used.

Kim I., Park C.S, & Lee H.Y. (2019). Angiotensin II Type 1 Receptor Blocker, Fimasartan, Reduces Vascular Smooth Muscle Cell Senescence by Inhibiting the CYR61 Signaling Pathway. Korean Circulation Journal, 49(7), 615-626.

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