Mouse skeletal muscle C2C12 cells were initially plated on 15 cm plates in DMEM with 20% fetal bovine serum. At confluence, the cells were switched to low serum medium to initiate myogenic differentiation. For extraction of total RNA, cells were first rinsed in PBS and then lysed in Trizol reagent (Invitrogen catalog # 15596-026) either during exponential growth in high serum medium, or at 60 hrs, 5 days and 7 days after medium shift. Residual contaminating genomic DNA was removed from the total RNA fraction using Turbo DNA-free (Ambion catalog # AM1907M). mRNA was isolated from DNA-free total RNA using the Dynabeads mRNA Purification Kit (Invitrogen catalog # 610-06).
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Myogenesis
Myogenesis
Myogenesis is the process of muscle cell development and differentiation.
It involves the formation of skeletal muscle fibers from progenitor cells, such as satellite cells and myoblasts.
This complex process is regulated by numerous signaling pathways and transcription factors that control cell proliferation, migration, and fusion.
Myogenesis is critical for the growth, repair, and maintenance of skeletal muscle tissue, and its dysregulation can contribute to various muscle-related disorders.
Understanding the molecular mechanisms underlying myogenesis is crucial for developing therapies to treat conditions affecting muscle structure and function.
It involves the formation of skeletal muscle fibers from progenitor cells, such as satellite cells and myoblasts.
This complex process is regulated by numerous signaling pathways and transcription factors that control cell proliferation, migration, and fusion.
Myogenesis is critical for the growth, repair, and maintenance of skeletal muscle tissue, and its dysregulation can contribute to various muscle-related disorders.
Understanding the molecular mechanisms underlying myogenesis is crucial for developing therapies to treat conditions affecting muscle structure and function.
Most cited protocols related to «Myogenesis»
Cells
Fetal Bovine Serum
Genome
Mus
Myogenesis
RNA, Messenger
Serum
Skeletal Myocytes
trizol
Mouse skeletal muscle C2C12 cells were initially plated on 15 cm plates in DMEM with 20% fetal bovine serum. At confluence, the cells were switched to low serum medium to initiate myogenic differentiation. For extraction of total RNA, cells were first rinsed in PBS and then lysed in Trizol reagent (Invitrogen catalog # 15596-026) either during exponential growth in high serum medium, or at 60 hrs, 5 days and 7 days after medium shift. Residual contaminating genomic DNA was removed from the total RNA fraction using Turbo DNA-free (Ambion catalog # AM1907M). mRNA was isolated from DNA-free total RNA using the Dynabeads mRNA Purification Kit (Invitrogen catalog # 610-06).
Cells
Fetal Bovine Serum
Genome
Mus
Myogenesis
RNA, Messenger
Serum
Skeletal Myocytes
trizol
Human myoblasts were isolated from biopsies and cultivated as described previously [19 (link)] in a growth medium consisting of 199 medium and DMEM (Invitrogen Carlsbad, CA) in a 1:4 ratio, supplemented with 20% FCS (Invitrogen), 2.5 ng/ml hepatocyte growth factor (Invitrogen), 0.1 μmol/l dexamethasone (Sigma-Aldrich, St. Louis, MO, USA) and 50 μg/ml gentamycin (Invitrogen). The myogenic purity of the populations was monitored by immunocytochemistry using desmin as marker. Enrichment of myogenic cells was performed using an immunomagnetic cell sorting system (MACS; Miltenyi Biotec, Paris, France) according to the manufacturer's instructions. Briefly, cells were labeled with anti-CD56 (a specific marker of myoblasts) microbeads, and then separated in a MACS column placed in a magnetic field. Purification was checked by immunochemistry using a desmin marker. Differentiation was induced at confluence by replacing the growth medium with DMEM supplemented with 100 μg/ml transferrin, 10 μg/ml insulin and 50 μg/ml of gentamycin (Sigma-Aldrich).
Biopsy
Cells
Culture Media
Desmin
Dexamethasone
Gentamicin
Hepatocyte Growth Factor
Homo sapiens
Immunocytochemistry
Insulin
Magnetic Fields
Microspheres
Myoblasts
Myogenesis
Population Group
Transferrin
Face
Myogenesis
Neurogenesis
Strains
Vision
Myobundles were formed by modifying our previously published methods for engineered rodent muscle tissues (Hinds et al., 2011 (link); Juhas et al., 2014 (link)) (Figure 1—figure supplement 2 ). Expanded myogenic cells were dissociated in 0.025% trypsin-EDTA to a single cell suspension and encapsulated in a fibrinogen (Akron, Boca Raton, FL) and matrigel solution on laser cut Cerex frames (9.2 × 9.5 mm outer dimensions, 6.8 × 8.3 mm inner dimensions) within PDMS molds (cast from Teflon masters and pretreated with pluronic) at 15 × 106 cells/ml (7.5 × 105 cells per myobundle). Specifically, a cell solution (7.5 × 105 cells in 17.2 µl media per bundle + 2 µl of 50 unit/ml thrombin in 0.1% BSA in PBS [Sigma, St. Louis, MO]) and a gelling solution (11 µl media + 10 µl Matrigel + 10 µl of 20 mg/ml Fibrinogen in DMEM) were prepared in separate vials on ice for up to six myobundles per vial. Gelling solution was added to the cell solution and mixed thoroughly then each bundle was individual pipetted within the PDMS mold and onto the frame. The cell/hydrogel mixture was polymerized for 30 min at 37°C followed by incubation in growth media containing 1.5 mg/ml 6-aminocaproic acid (ACA, Sigma). Myobundles were kept in growth media during gel compaction (3–5 days) and then switched to low glucose DMEM with 2% horse serum (Hyclone, Logan, UT), 2 mg/ml ACA and 10 µg/ml insulin (Sigma). Frames were removed from molds at the time of switch to low serum medium and cultured dynamically in suspension for an additional 1–4 weeks. Starting from a 50 mg donor biopsy, typical cell expansion for 5 passages can allow generation of at least 1000 myobundles with a total mass of >5 g, representing a >100-fold amplification of muscle mass when going from native to engineered tissue system.
All drugs were purchased from Sigma. Clenbuterol hydrochloride, chloroquine phosphate, and cerivastatin sodium salt hydrate were prepared at 1000× stock solutions in PBS (control) and sterile-filtered for use. Lovastatin was prepared as a 10,000× stock solution in DMSO in which case DMSO was used as vehicle control. Drugs studies in myobundles or 2D cultures were initiated after 1 week of differentiation. Myobundles were replenished with fresh media and drug each day to maintain drug concentration.
All drugs were purchased from Sigma. Clenbuterol hydrochloride, chloroquine phosphate, and cerivastatin sodium salt hydrate were prepared at 1000× stock solutions in PBS (control) and sterile-filtered for use. Lovastatin was prepared as a 10,000× stock solution in DMSO in which case DMSO was used as vehicle control. Drugs studies in myobundles or 2D cultures were initiated after 1 week of differentiation. Myobundles were replenished with fresh media and drug each day to maintain drug concentration.
(Z)-2-amino-5-chlorobenzophenonamidinohydrazone acetate
6-Aminocaproic Acid
Biopsy
Brown Oculocutaneous Albinism
CD3EAP protein, human
Cells
cerivastatin
chloroquine phosphate
Clenbuterol
Dietary Supplements
Edetic Acid
Equus caballus
Fibrinogen
Fungus, Filamentous
Glucose
Homo sapiens
Hydrogels
Insulin
Lovastatin
matrigel
Muscle Tissue
Myogenesis
Pharmaceutical Preparations
Pluronics
Reading Frames
Rodent
Serum
Sodium Chloride
Sodium Hydroxide
Sterility, Reproductive
Sulfoxide, Dimethyl
Teflon
Thrombin
Tissue Donors
Tissue Engineering
Trypsin
Most recents protocols related to «Myogenesis»
Satellite cell isolation and labelling were performed as described in Fabbrizio et al. [25 (link)]. Briefly, hindlimb muscles were isolated from sacrificed mice at 3 weeks and digested for 45 min at 37 °C under agitation in phosphate-buffered saline (PBS) (Sigma-Aldrich) supplemented with Dispase II (2.4 U/mL, Roche), Collagenase A (2 mg/ml, Roche), 0.4-mM CaCl2, 5-mM MgCl2, and deoxyribonuclease I (DNase I) (10 μg/mL, Roche). Cell suspensions were resuspended in HBSS and filtered with 100-μm and 40-μm filters. Single-cell suspension was stained with CD45/CD31/Ter119 phycoerythrin (PE) for lineage exclusion, Sca1 (Stem cell antigen 1)-fluorescein isothiocyanate (FITC) and α7 integrin allophycocyanin (APC). Cells were sorted using Moflo Astrios (Beckman Coulter). SCs were seeded on Matrigel-coated plates (Corning) at low-density (3500 cells/cm2) and cultured in Cyto-Grow (Resnova) complete medium as a growth medium (GM) for 4 days. For myogenic differentiation, after reaching the confluence, SCs have been shifted in DMEM + 2% horse serum up to 48 h. SC proliferation was evaluated for each well on stereological image fields acquired with an Olympus virtual slide system VS110 (Olympus) by counting the number of DAPI+/Ki67+ (Anti-Ki67 Ab; Abcam) cells per field. SC differentiation was assessed by evaluating fibre dimension and the fusion index (%) given by the number of nuclei per myotubes stained with anti-MyHC (DSHB).
allophycocyanin
Antigens
CASP3 protein, human
Cell Nucleus
Cells
Cell Separation
Collagenase
Culture Media
DAPI
Deoxyribonuclease I
dispase II
Equus caballus
Fibrosis
Fluorescein
Hemoglobin, Sickle
Hindlimb
Integrins
isolation
isothiocyanate
Magnesium Chloride
matrigel
Mus
Muscle Tissue
Myogenesis
Phosphates
Phycoerythrin
Saline Solution
Satellite Cell, Muscle
Serum
Skeletal Myocytes
Stem Cells
The sorted SCs populations were cultured in collagen I-coated dishes. The culture medium was F10 basic medium supplemented with 20% fetal bovine serum, 1% penicillin-streptomycin and 10 ng/mL basic fibroblast growth factor. The growth medium was changed every other day. The proliferation ability of SCs was evaluated using the BeyoClick EdU Cell Proliferation Kit following the manufacturer’s instructions. Briefly, EdU was added into the culture medium and incubated for 6 h. The cells were fixed and permeabilized and click additive solution and Hoechst 33,342 were added. The proliferation ability of SCs was calculated by dividing the number of EdU-positive cells by the total number of cells. To induce myogenic differentiation, 20,000 SCs/well were seeded in a 48-well plate coated with collagen I and cultured in a differentiation medium (2% horse serum and 1% penicillin-streptomycin in DMEM) for 48 h. The differentiation ability of SCs was evaluated using myotube staining. Briefly, myotubes were fixed with 4% PFA, permeabilized and blocked with 0.2% Triton X-100 and QuickBlock blocking buffer at room temperature, respectively. The myotubes were incubated with a primary antibody against MyHC (MF20) overnight at 4°C and subsequently incubated with the secondary antibody goat anti-mouse Alexa Fluor 568 at room temperature. The cells were counterstained with DAPI. The differentiation index was calculated by dividing the number of nuclei per myotube by the number of DAPI+ nuclei (all MyHC+ myotubes were considered). The fusion index was calculated by dividing the number of nuclei per myotube by the number of DAPI+ nuclei (only MyHC+ myotubes with two or more nuclei were considered).
alexa 568
Antibodies, Anti-Idiotypic
Buffers
Cell Nucleus
Cell Proliferation
Cells
Collagen Type I
Culture Media
DAPI
Equus caballus
Fetal Bovine Serum
Fibroblast Growth Factor 2
Goat
Hyperostosis, Diffuse Idiopathic Skeletal
Immunoglobulins
Mus
Myogenesis
Penicillins
Population Group
Serum
Skeletal Myocytes
Streptomycin
Triton X-100
Total muscle explant-derived cells were purified by Magnetic-activated cell sorting (Milteny Biotech) using anti-CD56 antibody (Table S1 ). Myoblast identity was determined by desmin immunostaining (> 98%). The primary and immortalized myoblasts were grown, respectively, in DMEM with high glucose and l -glutamine (Lonza), 10% fetal bovine serum (FBS) (Invitrogen), 1% Ultroser G (Pall BioSepra, Cergy-St-Christophe, France), and gentamicin (50 μg/ml, Sigma-Aldrich) or DMEM high glucose supplemented with 16.5% medium 199 (Lonza), 15% FBS, Ultroser G, HEPES 1 M (Sigma-Aldrich), zinc sulfate (Sigma ®-Aldrich, vitamin B12 (Sigma-Aldrich), and penicillin/streptomycin (pen/strep) at 37 °C under atmosphere with 5% CO2. For myogenic differentiation, cells were cultured on Matrigel-coated culture dishes and a differentiation medium was added after cells reached 100% confluence. This medium was composed of DMEM/gentamicin (50 μg/ml) with 2% FBS for primary cells and DMEM high glucose, medium 199 supplemented with 0.5% insulin, 1% apo-transferrin (Sigma-Aldrich), 2% HEPES 1 M and pen/strep for immortalized cells. HEK293 were grown in DMEM high glucose-10% FBS and pen/strep. Transfection of primary cells was previously reported [3 ]. The KLF15 expression vector was a generous gift of Prof. Yegor Vassetzky [18 (link)].
Antibodies, Anti-Idiotypic
Atmosphere
Cells
Cloning Vectors
Cobalamins
Desmin
Fetal Bovine Serum
Gentamicin
Glucose
Glutamine
HEPES
Hyperostosis, Diffuse Idiopathic Skeletal
Insulin
matrigel
Muscle Cells
Myoblasts
Myogenesis
Penicillins
Streptomycin
Transfection
Transferrin
Ultroser G
Zinc Sulfate
Protocol full text hidden due to copyright restrictions
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Animals
Animals, Laboratory
Biological Assay
Gene Activation
Lanugo
Males
Mus
Muscle Tissue
Myogenesis
Myogenin
OGG1 protein, human
Protocol full text hidden due to copyright restrictions
Open the protocol to access the free full text link
Atmosphere
Caimans
Cells
Equus caballus
Fetal Bovine Serum
Fibroblasts
Glutamine
Guanine
Myoblasts
Myogenesis
Penicillins
Serum
Streptomycin
Top products related to «Myogenesis»
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Fetal Bovine Serum (FBS) is a cell culture supplement derived from the blood of bovine fetuses. FBS provides a source of proteins, growth factors, and other components that support the growth and maintenance of various cell types in in vitro cell culture applications.
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DMEM (Dulbecco's Modified Eagle's Medium) is a cell culture medium formulated to support the growth and maintenance of a variety of cell types, including mammalian cells. It provides essential nutrients, amino acids, vitamins, and other components necessary for cell proliferation and survival in an in vitro environment.
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Horse serum is a biological fluid derived from the blood of horses. It contains a complex mixture of proteins, including immunoglobulins, hormones, and other biomolecules. Horse serum is commonly used as a supplement in cell culture media to support the growth and maintenance of various cell types.
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Penicillin/streptomycin is a commonly used antibiotic solution for cell culture applications. It contains a combination of penicillin and streptomycin, which are broad-spectrum antibiotics that inhibit the growth of both Gram-positive and Gram-negative bacteria.
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Dexamethasone is a synthetic glucocorticoid medication used in a variety of medical applications. It is primarily used as an anti-inflammatory and immunosuppressant agent.
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Lipofectamine 2000 is a cationic lipid-based transfection reagent designed for efficient and reliable delivery of nucleic acids, such as plasmid DNA and small interfering RNA (siRNA), into a wide range of eukaryotic cell types. It facilitates the formation of complexes between the nucleic acid and the lipid components, which can then be introduced into cells to enable gene expression or gene silencing studies.
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DMEM/F12 is a cell culture medium developed by Thermo Fisher Scientific. It is a balanced salt solution that provides nutrients and growth factors essential for the cultivation of a variety of cell types, including adherent and suspension cells. The medium is formulated to support the proliferation and maintenance of cells in vitro.
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TRIzol reagent is a monophasic solution of phenol, guanidine isothiocyanate, and other proprietary components designed for the isolation of total RNA, DNA, and proteins from a variety of biological samples. The reagent maintains the integrity of the RNA while disrupting cells and dissolving cell components.
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L-glutamine is an amino acid that is commonly used as a dietary supplement and in cell culture media. It serves as a source of nitrogen and supports cellular growth and metabolism.
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The RNeasy Mini Kit is a laboratory equipment designed for the purification of total RNA from a variety of sample types, including animal cells, tissues, and other biological materials. The kit utilizes a silica-based membrane technology to selectively bind and isolate RNA molecules, allowing for efficient extraction and recovery of high-quality RNA.
More about "Myogenesis"
Myogenesis: The Key to Unlocking Muscle Development and Regeneration Myogenesis, the intricate process of muscle cell formation and differentiation, is a critical aspect of skeletal muscle growth, repair, and maintenance.
This complex biological pathway involves the transformation of progenitor cells, such as satellite cells and myoblasts, into fully functional muscle fibers.
Understanding the intricacies of myogenesis is crucial for unraveling the mysteries of various muscle-related disorders and developing effective therapies.
Numerous signaling pathways and transcription factors orchestrate the delicate balance of cell proliferation, migration, and fusion during this process.
Researchers investigating myogenesis often utilize a range of cell culture media and supplements to facilitate the optimal development of muscle cells in vitro.
Fetal Bovine Serum (FBS), Dulbecco's Modified Eagle Medium (DMEM), Horse Serum, Penicillin/Streptomycin, and Dexamethasone are commonly employed to support the growth and differentiation of muscle progenitor cells.
Additionally, techniques like Lipofectamine 2000 transfection, DMEM/F12 culture media, TRIzol reagent extraction, and L-glutamine supplementation play crucial roles in studying gene expression, RNA isolation, and other molecular mechanisms underlying myogenesis.
By leveraging the insights gained from the MeSH term description and the Metadescription, researchers can streamline their myogenesis studies, identify the best protocols and products, and ultimately contribute to the advancement of muscle-related therapies.
PubCompare.ai, an AI-driven platform, can be a valuable tool in this endeavor, providing a seamless and efficient way to navigate the vast landscape of myogenesis research.
This complex biological pathway involves the transformation of progenitor cells, such as satellite cells and myoblasts, into fully functional muscle fibers.
Understanding the intricacies of myogenesis is crucial for unraveling the mysteries of various muscle-related disorders and developing effective therapies.
Numerous signaling pathways and transcription factors orchestrate the delicate balance of cell proliferation, migration, and fusion during this process.
Researchers investigating myogenesis often utilize a range of cell culture media and supplements to facilitate the optimal development of muscle cells in vitro.
Fetal Bovine Serum (FBS), Dulbecco's Modified Eagle Medium (DMEM), Horse Serum, Penicillin/Streptomycin, and Dexamethasone are commonly employed to support the growth and differentiation of muscle progenitor cells.
Additionally, techniques like Lipofectamine 2000 transfection, DMEM/F12 culture media, TRIzol reagent extraction, and L-glutamine supplementation play crucial roles in studying gene expression, RNA isolation, and other molecular mechanisms underlying myogenesis.
By leveraging the insights gained from the MeSH term description and the Metadescription, researchers can streamline their myogenesis studies, identify the best protocols and products, and ultimately contribute to the advancement of muscle-related therapies.
PubCompare.ai, an AI-driven platform, can be a valuable tool in this endeavor, providing a seamless and efficient way to navigate the vast landscape of myogenesis research.