Cryopreserved primary human myocytes (Lonza CC-2561) were thawed, centrifuged and resuspended in complete media (SkGM-2 Bullet kit, Lonza) following the manufacturer's directions. Cells were plated in Laminin-coated plates (Corning) and allowed to adhere and grow to 90% confluence. Media was changed to basal media containing 2% horse serum (Life Technologies, Inc.) and 5 μg ml−1 insulin, and cells continued in culture until myotubes formed. For sterol synthesis assays, fresh basal media containing [14C]-glucose, and vehicle, ETC-1002, simvastatin, or atorvastatin was added to myotube cultures for an additional 12 h. Viability after 12 h was assessed in parallel cultures using MTT assay, and non-saponifiable lipids were extracted as described below. Morphology was determined by visual assessment of merged images from bright-field and fluorescent capture ( × 20) of Hoechst-stained cultured primary human myotubes treated with vehicle, statins (±500 μM mevalonate) or ETC-1002 (100 μM) for 48 h. Viability/cytotoxicity was determined after 48 h treatment by measuring GF-AFC/bis-AAF-R110 cleavage and Caspase 3, 7 activity luciferase-based DEVD cleavage (ApoTox-Clo, Promega).
Laminin coated plates
Manufactured by Corning
Laminin-coated plates are a type of lab equipment designed for cell culture applications. Laminin, a naturally occurring protein, is coated onto the surface of the plates to provide a favorable extracellular matrix for the adhesion, growth, and differentiation of various cell types.
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Lab products found in correlation
Horse serum, by Thermo Fisher Scientific (1 mentions)
Cc 2561, by Lonza (1 mentions)
Skgm 2 bullet kit, by Lonza (1 mentions)
Rencell vm, by Merck Group (1 mentions)
Human epidermal growth factor egf, by Merck Group (1 mentions)
B27 neural supplement, by Thermo Fisher Scientific (1 mentions)
Heparin, by STEMCELL (1 mentions)
2 protocols using laminin coated plates
1
Myotube Viability and Lipid Synthesis Assay
2
Differentiation of ReNcell VM Neural Progenitors
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The immortalized human ReNcell VM (Ventral Mesencephalon) neural progenitor cells (EMD Millipore) were cultured on 20 μg/ml laminin-coated plates (Corning), in Dulbecco’s Modified Eagle Medium (DMEM): Nutrient Mixture F12 (DMEM/F12) media (Life Technologies) supplemented with 2% (v/v) B27 neural supplement (Life Technologies), 2 μg/ml heparin (StemCell Technologies), 20 μg/ml human Epidermal Growth Factor (EGF; Sigma), 20 μg/ml human Fibroblast Growth Factor-basic (bFGF; Life Technologies), and 100 units/ml penicillin-streptomycin solution (GE Hyclone). The culture media was changed every 3 days until the cells were confluent and ready for subculturing. Differentiation of the neural progenitor cells was initiated by replacing the culture media with DMEM/F12 supplemented with 2% (v/v) B27 neural supplement, 2 μg/ml heparin, and 100 units/ml penicillin-streptomycin solution without the growth factors EGF and bFGF. The differentiation media was changed every 3 days for 3–4 weeks. All cells were grown in 5% CO2 at 37 °C.
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