Cc 2580

Manufactured by Lonza

Sourced in United States, United Kingdom

The CC-2580 is a laboratory equipment product designed for general use. It serves as a tool for conducting various experiments and analyses in a controlled laboratory setting. The core function of the CC-2580 is to provide a reliable and consistent platform for research and testing activities.

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Lab products found in correlation

23 protocols using cc 2580

Primary Myoblast Differentiation Protocol

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Reagents were obtained from Sigma (Gillingham, UK) unless stated otherwise. Primary myoblasts (CC‐2580; Lonza, Slough, UK) were maintained in house in Skeletal Muscle Basal Medium‐2 (Lonza; CC‐3244 and CC‐3246) containing 0.1% human epidermal growth factor, 2% l‐glutamine, 10% fetal bovine serum (FBS) and 0.1% gentamicin/amphotericin‐B in the absence of GCs. Confluent myotubes were differentiated in Dulbecco's modified Eagle's medium (DMEM) containing 2% horse serum (HS) for 120 h. Media were replaced every 2–3 days 25.

Hardy R.S., Doig C.L., Hussain Z., O'Leary M., Morgan S.A., Pearson M.J., Naylor A., Jones S.W., Filer A., Stewart P.M., Buckley C.D., Lavery G.G., Cooper M.S, & Raza K. (2016). 11β‐Hydroxysteroid dehydrogenase type 1 within muscle protects against the adverse effects of local inflammation. The Journal of Pathology, 240(4), 472-483.

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Culturing and Treating Rhabdomyosarcoma Cell Lines

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RMS cell lines (CW9019, RD, and RH4) were kindly provided by Dr. Oscar Martínez-Tirado and Dr. Cristina Muñoz-Pinedo from the Bellvitge Biomedical Research Institute (IDIBELL). C2C12 cells were purchased at ATCC (ATCC^® CRL-1772^™, ATCC, Manassas, VI, USA). Human skeletal muscle myoblasts (HSMM) were purchased at Lonza (CC-2580, Lonza, Basel, Switzerland). RMS cell lines were cultured in RPMI 1640 medium (31870, Thermo Fisher, Gibco, Paisley, Scotland) supplemented with 10% heat-inactivated fetal bovine serum (10270, Thermo Fisher, Gibco), 1% of l-glutamine (25030, Thermo Fisher, Gibco), and 1% of penicillin and streptomycin (15140, Thermo Fisher, Gibco). C2C12 cells were cultured in DMEM high glucose medium (41965, Thermo Fisher, Gibco) supplemented with 10% heat-inactivated fetal bovine serum (10270, Thermo Fisher, Gibco) and 1% of penicillin and streptomycin (15140, Thermo Fisher, Gibco). HSMM cells were cultured in SKBM-2 medium (CC-3246, Lonza) supplemented with its specific SingleQuots^TM and growth factors (CC-3244, Lonza). All cells were tested for mycoplasma and maintained at 37 °C in a humidified atmosphere of 5% CO₂. Drug treatments were performed directly in the culture media at the doses and time points indicated in every single experiment. All drugs were purchased at Selleckchem (Munich, Germany).

Alcon C., Manzano-Muñoz A., Prada E., Mora J., Soriano A., Guillén G., Gallego S., Roma J., Samitier J., Villanueva A, & Montero J. (2020). Sequential combinations of chemotherapeutic agents with BH3 mimetics to treat rhabdomyosarcoma and avoid resistance. Cell Death & Disease, 11(8), 634.

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Cell Culture Protocol for Rhabdomyosarcoma Models

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aRMS cell line Rh30, eRMS cell lines RD and Rh18, and PDX cultures PCB380 and PCB82 were cultured in growth medium (GM) RPMI 1640 (11875-093; Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine Serum (FBS) (26140079; Thermo Fisher Scientific) and 1% penicillin/Streptomycin (15140-122; Thermo Fisher Scientific). Primary human skeletal myoblasts (HSMM) (CC-2580; Lonza Inc, Allendale, NJ, USA) were cultured in GM (SKBM-2 CC-3244; Lonza Inc) supplemented with 10% FBS and 1% penicillin/Streptomycin. HSMM was differentiated until day 4 in differentiation medium DM: F-12 (11320033; Thermo Fisher Scientific). All cells were incubated at 37°c and 5% CO₂. aRMS and eRMS cell lines were authenticated by short tandem repeat (STR) analysis (Biosynthesis, Lewisville, TX, USA).

Bharathy N., Svalina M.N., Settelmeyer T.P., Cleary M.M., Berlow N.E., Airhart S.D., Xiang S., Keck J., Hayden J.B., Shern J.F., Mansoor A., Lathara M., Srinivasa G., Langenau D.M, & Keller C. (2017). Preclinical testing of the glycogen synthase kinase-3β inhibitor tideglusib for rhabdomyosarcoma. Oncotarget, 8(38), 62976-62983.

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Skeletal Muscle Myoblast Differentiation

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Human skeletal muscle myoblasts (hSKmc) (CC-2580), growth media, supplements, and serum (CC-3245) were purchased from Lonza (Walkersville, MD). HSMM differentiation medium (DM) was prepared by adding 2% horse serum to DMEM (Hyclone). HSMM were maintained in growth media for 3–4 days till cultures reached 70% confluence followed by three washes in phosphate buffered saline (PBS, pH 7) and switched to DM. Intracellular metabolites from cell lysates were collected from Day 0 cultures (before addition of DM), Day 2 and Day 4 cultures (DM added for 2 and 4 days respectively). For kinetic analysis of intracellular metabolites, hSKmc cultures for Day 0, 2, and 4 underwent three washes in PBS, scraped in RIPA lysis and extraction buffer (Cat # 89900, ThermoFisher Scientific), and stored at – 80 °C.

Kumar A., Kumar Y., Sevak J.K., Kumar S., Kumar N, & Gopinath S.D. (2020). Metabolomic analysis of primary human skeletal muscle cells during myogenic progression. Scientific Reports, 10, 11824.

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Generating Innervated Neuromuscular Junctions

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Primary human SkM myoblasts (hSMM) were obtained from Lonza, Allendale, NJ, USA, from a healthy donor (Lonza CC2580, Lot No. 8TL180368), passaged once in serum-free Adult Growth Medium (AGM) [22 (link)] (Figure S4c), and cryopreserved at 60 – 70% confluency. Cryopreserved P1 hSMM were thawed and seeded along the microtunnels of the NMJ systems at a density of 300 cells/mm² in AGM. Myoblasts were proliferated to 85% confluency and switched to a serum-free differentiation medium (NBActiv4, Brainbits) supplemented with 1% Antibiotic-Antimycotic (Thermo Fisher 15240–062) to promote fusion into myotubes. Cultures were maintained at 37°C and 5% CO₂ with full medium changes every two days.
Myotube fusion was typically observed after 48 hours of incubation in differentiation medium, at which point differentiated iPSC-hMNs were seeded into the laminin-coated chamber opposite the SkM chamber with hMN maturation medium (Figure S4b). MNs and SkM were allowed to mature in the adjacent chambers for 10 to 12 days, during which the iPSC-hMNs extended axons through microtunnels in the PDMS barrier and innervated the fused myotubes. NMJ systems were maintained at 37°C and in 5% CO₂ with full medium changes using hMN maturation medium and Nba4 every two days, functionally evaluated on NMJ day 12, and fixed for immunocytochemical analysis immediately after testing.

Sasserath T., Robertson A.L., Mendez R., Hays T.T., Smith E., Cooper H., Akanda N., Rumsey J.W., Guo X., Farkhondeh A., Pradhan M., Baumgaertel K., Might M., Rodems S., Zheng W, & Hickman J.J. (2022). An induced pluripotent stem cell-derived NMJ platform for study of the NGLY1-Congenital Disorder of Deglycosylation. Advanced therapeutics, 5(11), 2200009.

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Establishment and Maintenance of ERMS Cell Lines

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Three ERMS cell line stocks were provided by JCRB Cell Bank (National Institutes of Biomedical Innovation, Health and Nutrition, Osaka, Japan): ERMS1 cells (JCRB1648) derived from the anaplastic pelvic ERMS of a 5-year-old female [5 (link)], KYM1 cells (JCRB0627) derived from the neck ERMS of a 9-month-old infant [28 (link)], and RD cells (JCRB9072) derived from the malignant pelvic ERMS of a 7-year-old female [29 (link)]. The ERMS cells were maintained in RPMI1640 (2 g/L glucose) (Nacalai, Osaka, Japan) with 10% fetal bovine serum (HyClone; GE Healthcare, Salt Lake City, UT, USA), 100 units/mL penicillin, and 100 μg/mL streptomycin at 37 °C with 5% CO₂ [30 (link)]. The commercially available human myoblasts isolated from the healthy subject (CC-2580; Lonza, Walkersville, MD, USA) were maintained in Skeletal Muscle Growth Media-2 (CC-3245; Lonza) on the dishes coated with collagen type I-C (Cellmatrix; Nitta Gelatin, Osaka, Japan) at 37 °C with 5% CO₂ [16 (link),17 (link),19 (link),31 (link)].

Nohira N., Shinji S., Nakamura S., Nihashi Y., Shimosato T, & Takaya T. (2022). Myogenetic Oligodeoxynucleotides as Anti-Nucleolin Aptamers Inhibit the Growth of Embryonal Rhabdomyosarcoma Cells. Biomedicines, 10(11), 2691.

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Myoblast Differentiation and Cytokine Treatment

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The commercially available human myoblast stock isolated from healthy subject (CC-2580; Lonza, MD, USA) was used [14] (link)[15] (link)[16] (link)18] (link). The myoblasts were cultured on dishes coated with collagen type I-C (Cellmatrix; Nitta Gelatin, Osaka, Japan) at 37°C under 5% CO2. Undifferentiated myoblasts were maintained in Skeletal Muscle Growth Media-2 (CC-3245; Lonza). 7.5 × 10 4 myoblasts were seeded on 30-mm dishes for immunocytochemistry, and 1.5 × 10 5 myoblasts were seeded on 60-mm dishes for quantitative real-time PCR (qPCR). The next day, myogenic differentiation was induced by replacing the medium with differentiation medium (DM) consisting of DMEM supplemented with 2% horse serum (GE Healthcare) and PS. For CM experiments, CM was added at a final concentration of 30-50%. Non-supplemented DMEM was used as a negative control for CM. For TNF-α experiments, the myoblasts were pre-treated with 30 μM iSN04 for 3 h and then treated with 10 ng/ml TNF-α.

Nihashi Y., Yamamoto M., Shimosato T., & Takaya T. (2021). Myogenetic oligodeoxynucleotide restores differentiation and reverses inflammation of myoblasts aggravated by cancer-conditioned medium.

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Differentiation of Primary Myoblasts

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Reagents were obtained from Sigma (Gillingham, UK) unless stated otherwise. Primary myoblasts obtained from healthy donors (CC‐2580; Lonza, Slough, UK) were maintained in-house in Skeletal Muscle Basal Medium‐2 (Lonza; CC‐3244 and CC‐3246) containing 0.1% human epidermal growth factor, 2% l‐glutamine, 10% fetal bovine serum (FBS), and 0.1% gentamicin/amphotericin‐B in the absence of GCs. Confluent myotubes were differentiated in Dulbecco’s modified Eagle’s medium (DMEM) containing 2% horse serum (HS) for 120 h. Media were replaced every 2–3 days as previously reported (16 (link)).

Webster J.M., Waaijenberg K., van de Worp W.R., Kelders M.C., Lambrichts S., Martin C., Verhaegen F., Van der Heyden B., Smith C., Lavery G.G., Schols A.M., Hardy R.S, & Langen R.C. (2023). 11β-HSD1 determines the extent of muscle atrophy in a model of acute exacerbation of COPD. American Journal of Physiology - Lung Cellular and Molecular Physiology, 324(4), L400-L412.

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Cell Culture Protocols for Various Cell Lines

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HEK293T (ATCC CRL-3216™) and HEK293T-derived CLCN5 GFP reporter cells, described recently (42 (link)), were cultured in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum (FBS), 2 mM l-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin (Thermo Fisher Scientific) at 37°C in an incubator with 5% CO₂. Human IMR90 cells (lung fibroblasts; ATCC CCL-186) were cultured in Eagle’s minimum essential medium supplemented with 10% FBS, 2 mM l-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin. Human CD34⁺ progenitor cells from mobilized peripheral blood (Lonza, catalog # 4Y-101C) were cultured in serum-free medium (Stem Cell Technology, catalog # 09605) supplemented with 1× StemSpan™ CD34⁺ Expansion Supplement (Stem Cell Technology, catalog # 02691). Human skeletal muscle myoblasts (Lonza, catalog # CC-2580) were cultured with an SkGM™-2 Skeletal Muscle Cell Growth Medium-2 BulletKit™ (Lonza, catalog # CC-3245).

Yoo K.W., Yadav M.K., Song Q., Atala A, & Lu B. (2022). Targeting DNA polymerase to DNA double-strand breaks reduces DNA deletion size and increases templated insertions generated by CRISPR/Cas9. Nucleic Acids Research, 50(7), 3944-3957.

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Isolation and Culture of Muscle Precursor Cells

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Rat muscle precursor cells (MPCs) were isolated similar to that described previously [28 (link)]. Briefly, skeletal muscles (tibialis anterior, extensor digitorum longus, quadriceps, gastrocnemius, and plantaris) were collected from each rat, minced, digested with protease type IV, and filtered with a 100 μm Steriflip to remove debris. Harvested cells were plated on a tissue culture-treated plate for 12 hours. Subsequently, non-adherent MPCs were transferred to a Matrigel-coated dishes, and MPCs were then cultured in growth medium (GM) composed of 20% fetal bovine serum (FBS), 1% penicillin-streptomycin (P/S), 0.2% MycoZap (MZ), Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 1 g/L D-glucose, L-glutamine, and 110 mg/L sodium pyruvate. Human MPCs (HSMM; muscle myoblasts (CC-2580) or D-HSMM (CC2901)) were purchased, (Lonza, Basel, Switzerland) and cultured similarly to that described for rat MPCs.

Acosta F.M., Pacelli S, & Rathbone C.R. (2023). Diabetes diminishes muscle precursor cell-mediated microvascular angiogenesis. PLOS ONE, 18(8), e0289477.

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