Myogenesis: Methods and Protocols

Preface......Page 5
Contents......Page 6
Contributors......Page 8
 1 Introduction......Page 12
  2.1 Dissociation of Primary Cells......Page 13
   2.2.1 Thawing of Cryopreserved Sample......Page 14
   2.3.1 In Vitro Cell Culture......Page 15
  3.1 Dissociation of Primary Cells (See Note 3)......Page 16
   3.2.1 Thawing of Cryopreserved Sample Prior to FACS......Page 18
   3.2.2 Preparation of Sample for FACS......Page 19
   3.3.1 In Vitro Cell Culture......Page 20
   3.3.2 Cell Passaging......Page 21
   3.3.5 Immunofluorescent Staining for PAX7 (See Note 11)......Page 22
 4 Notes......Page 24
 References......Page 26
 1 Introduction......Page 27
 2 Materials......Page 28
 3 Methods......Page 29
  3.1 Establishment of Differentiated Human Skeletal Muscle Cells (Myotubes)......Page 30
   3.3.1 Cleaning......Page 31
 4 Notes......Page 32
 References......Page 34
 1 Introduction......Page 35
  2.4 Production of gRNAs by In Vitro Transcription......Page 39
  3.1 Generation of Cas9-Expressing C2C12 Cells......Page 40
  3.2 gRNA Selection (See Note 4)......Page 41
  3.3 MyoD KO Cell Line Selection (See Note 8)......Page 42
  3.5 Adipogenic Induction of MyoD KO Cell Line......Page 43
 4 Notes......Page 44
 References......Page 49
 1 Introduction......Page 52
 2 Materials......Page 53
  3.1 Chromatin Crosslinking and Cell Harvesting......Page 56
  3.2 Sonication......Page 57
  3.3 Immunoprecipitation......Page 58
  3.5 DNA Isolation......Page 59
 4 Notes......Page 60
 References......Page 62
 1 Introduction......Page 64
  2.2 Human Primary Cell Isolation......Page 66
  2.6 Fibrin Skeletal Muscle Constructs......Page 67
  2.7 TC-3 Bioreactor System (Ebers Medical Technology, ESP) (See Note 15)......Page 68
  3.1 Skeletal Muscle Biopsy Procedure......Page 69
  3.2 Human Primary Cell Isolation......Page 71
  3.3 Cryopreservation of Cells......Page 72
  3.5 Preparation of 3-D Culture Dishes......Page 73
  3.6 Preparation and Maintenance of Fibrin Skeletal Muscle Constructs......Page 75
  3.9 Electrical and Mechanical Stimulation......Page 77
 4 Notes......Page 81
 References......Page 87
 1 Introduction......Page 89
  2.1 Experimental Animals......Page 92
  2.2 General Equipment......Page 94
  2.5 Real-Time RT-qPCR......Page 95
  3.1 Satellite Cell Isolation......Page 96
  3.2 Percoll Density Gradient Centrifugation and Plating......Page 97
  3.3 Real-Time RT-qPCR......Page 98
 4 Notes......Page 99
 References......Page 100
 1 Introduction......Page 102
   2.1.1 Preparation and Identification of shRNA Plasmids......Page 104
   2.1.3 Knockdown In Vitro-C2C12 Cells......Page 105
   2.1.4 Knockdown In Vitro-Mouse Primary Myoblasts (In Addition to Materials Listed Above)......Page 106
   3.1.1 Preparation and Identification of shRNA Plasmids......Page 107
   3.1.2 Virus Production......Page 108
   3.1.3 Knockdown In Vitro-C2C12 Cells......Page 109
   3.1.4 Knockdown In Vitro-Mouse Primary Myoblasts......Page 111
   3.2.2 Tibialis Anterior (TA) Muscle Injury......Page 113
   3.2.4 Knockdown Confirmation-Immunohistochemistry......Page 114
 4 Notes......Page 115
 References......Page 117
 1 Introduction......Page 118
  2.1 Bovine Stromal Vascular Cell Isolation and Cell Culture......Page 120
   2.2.1 Lipid Accumulation Visualization with Oil Red O......Page 121
   2.3.1 Lipid Accumulation Visualization Within 3D Scaffolds......Page 122
  3.1 Bovine Stromal Vascular Cell Isolation......Page 123
   3.1.1 Maintenance of Stromal Vascular Cells in Culture......Page 124
   3.2.1 Lipid Accumulation Visualization with Oil Red O......Page 126
  3.3 Alginate Scaffolds for 3D Bovine Adipose Tissue Culture......Page 127
   3.3.1 Lipid Accumulation Visualization Within 3D Scaffolds......Page 128
 4 Notes......Page 129
 References......Page 132
 1 Introduction......Page 133
  2.2 Equipment and Materials for Microarray......Page 136
  3.3 RNA Extraction Protocol......Page 138
  3.5 Workflow for Sample Preparation and Two-Color Microarray Processing......Page 139
  3.6 Statistical Analysis of Microarray Data......Page 146
  3.8 Ontological Analysis Using PANTHER Classification System......Page 149
  3.9 Functional Gene Annotation Using DAVID Program......Page 153
  3.10 Analysis and Visualization of Gene Interactions Using Pathway Studio (See Note 49)......Page 155
  3.11 Validation of Microarray Data-Further Biological Analyses......Page 161
 4 Notes......Page 162
 References......Page 173
 1 Introduction......Page 175
 2 Materials......Page 177
  3.1 Coating of Six-Well Plate with Sylgard Silicone Elastomer......Page 178
  3.2 Isolation of Myoblasts from Human Biopsy (See Note 2)......Page 179
  3.4 Human Muscle Biopsy Characterization: Determination of Myoblast Fusion Index......Page 181
  3.5 Human Muscle Biopsy Characterization: Determination of Doubling Time......Page 182
  3.7 Production of Six Wells with Silicone Molds and Attachment Sites......Page 183
 4 Notes......Page 185
 References......Page 188
 1 Introduction......Page 190
  2.1 Tissue Culture and Cell Counting......Page 192
  2.4 Immunofluorescent Staining......Page 194
  3.1 Plate Preparation......Page 196
  3.3 C2C12 Myoblast Harvest and Seeding for Serum Free Experiments......Page 197
  3.4 Proliferation Assays......Page 198
  3.5 Differentiation Assays......Page 199
  3.6 Measuring Myotube Width......Page 201
   3.7.1 RNA Extraction......Page 203
   3.7.2 cDNA Synthesis......Page 204
    Primer Efficiency......Page 205
    Gene Expression Analyses......Page 206
   3.8.2 Seeding and Staining Cells......Page 207
 4 Notes......Page 212
 References......Page 216
 1 Introduction......Page 218
  2.1 C2C12 Cell Growth and Differentiation......Page 220
  2.3 Extraction Methods......Page 222
  2.5 Lipids, ACoA, and ACar Identification and Quantification......Page 223
   3.1.3 Lipid Extraction......Page 224
   3.2.4 Homogenization of Cells......Page 225
  3.3 Extraction of Acyl-CoA Esters and Acyl-Carnitines from Mouse Skeletal Muscle......Page 226
   3.4.3 Extraction of Acyl-CoA Esters and Acyl-Carnitines......Page 227
  3.6 LC-MS Conditions for ACoA and ACar Analysis......Page 228
  3.7 Targeted Identification and Quantification of Analytes......Page 229
 4 Notes......Page 230
 References......Page 232
 1 Introduction......Page 234
  2.2 C2C12 Seeding and Culture......Page 236
  2.5 Immunostaining......Page 237
  2.9 Real Time Quantitative PCR......Page 238
  3.2 siRNA Transfection of C2C12 Cells......Page 239
  3.3 Immunostaining......Page 240
  3.5 Image Analyses......Page 241
  3.6 Classification of Observed Phenotypes......Page 243
  3.8 Reverse Transcription......Page 244
 4 Notes......Page 245
 References......Page 246
 1 Introduction......Page 249
  2.3 Cell Dissociation and FACS Sorting......Page 250
  3.2 Cell Dissociation and FACS Sorting......Page 252
 4 Notes......Page 254
 References......Page 258
 1 Introduction......Page 259
  2.1 Bovine Primary Skeletal Muscle Cell Isolation......Page 261
  2.5 Peptide Generation......Page 262
  3.1 Bovine Primary Muscle Cell Isolation......Page 263
  3.2 Determination of Purity of the Isolated Muscle Cells......Page 264
  3.4 Protein Assay for Samples Containing Detergents......Page 265
  3.6 Protein Assay for Peptide Samples......Page 266
  3.7 Desalting of Peptide Samples......Page 267
 4 Notes......Page 268
 References......Page 270
 1 Introduction......Page 271
  2.2 Freezing Flies and Cryosectioning......Page 273
  3.1 Freezing Samples for Cryosectioning and Microsampling......Page 274
  3.3 Microsampling Cryosections......Page 276
  3.4 Adult Muscle Microdissection......Page 279
  3.5 Preparation of Dissected Samples for Protein Analysis......Page 281
 4 Notes......Page 282
 References......Page 284
 1 Introduction......Page 286
  2.1 Experimental Setup......Page 289
  2.3 Single Fiber Isolation and Processing......Page 290
  3.1 Experimental Setup......Page 291
  3.2 Muscle Dissection and Incubation......Page 293
  3.4 Measuring Glucose Uptake......Page 294
  3.5 MHC Isoform Identification......Page 295
 4 Notes......Page 297
 References......Page 302
 1 Introduction......Page 304
   2.2.1 For TiO2 Microtips Method......Page 307
   3.1.1 Lysis Method 1......Page 308
   3.1.2 Lysis Method 2......Page 309
  3.2 Sample Preparation for LC-MS/MS......Page 310
   3.2.1 Method 1: TiO2 Microtips......Page 311
  3.3 LC-MS/MS......Page 312
  3.5 Label-Free Peptide Quantification......Page 314
 4 Notes......Page 316
 References......Page 318
 1 Introduction......Page 321
  2.1 Atlantic Salmon Primary Muscle Cell Isolation......Page 323
  2.4 Stimulation of Atlantic Salmon Muscle Cell Differentiation......Page 325
  2.5 Trans-Differentiation of Atlantic Salmon Myosatellite Cells......Page 326
  3.1 Atlantic Salmon Primary Muscle Cell Isolation......Page 327
  3.5 Trans-Differentiation of Atlantic Salmon Myosatellite Cells......Page 329
 4 Notes......Page 330
 References......Page 331
 1 Introduction......Page 333
  2.1 Fly Strains and Large Fly Collections......Page 334
  2.3 Reagents for Muscle Morphology Assays......Page 335
  3.2 Fly Crosses for Muscle-Specific RNAi......Page 340
  3.3 High-Throughput Assays After RNAi-Based Gene Knock-Down: Lethality, Locomotion, and Flight Tests......Page 341
   3.4.2 Pupal or Adult Muscles......Page 342
  3.5 Low-Throughput Live Assays After RNAi-Based Gene Knock-Down: Myoblast Fusion, Myotube Attachment, Myofibrillogenesis, and .........Page 346
 4 Notes......Page 347
 References......Page 348
Index......Page 351