To monitor intracellular trafficking of fluorescent fusion proteins, C2C12 cells or mg53−/− myotubes were cultured in glass-bottom dishes (Bioptechs) and transfected with plasmid DNA using standard techniques. For immunocytochemistry, FDB fibres were fixed using 100% ethanol at −20 °C for 5 min before anti-mouse MG53 rabbit polyclonal antibody was applied at a 1:200 dilution. Cells were washed and secondary antibodies coupled with fluorescent probes (goat anti-rabbit Alexa Fluor 488 or Alexa Fluor 546) were applied according to the manufacturer’s instructions (Molecular Probes). For the assay of acute live-cell membrane damage, transfected cells were mechanically damaged using a micropipette attached to a micromanipulator. Fluorescence images were captured using a BioRad 2100 Radiance laser scanning confocal microscope with a ×40 1.3NA oil immersion objective.
Protocol detail
Tracking Intracellular Protein Trafficking in Myotubes
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A a 1
Alexa fluor 488
Alexa fluor 546
Anti antibody
Antibodies
Assay
Cell membrane
Cells
Confocal laser scanning microscope
Dilution
Dishes
Ethanol
Fibres
Fluorescence
Fluorescent probes
Goat
Immersion
Immunocytochemistry
Intracellular
Molecular probes
Mouse
Myotubes
Plasmid
Rabbit
Corresponding Organization :
Other organizations : Johnson University, Tohoku University, Tokyo Metropolitan Institute of Medical Science, Saitama Medical University
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Variable analysis
independent variables
- Transfection of plasmid DNA into C2C12 cells or mg53-/- myotubes
- Mechanical damage to transfected cells using a micropipette
- Intracellular trafficking of fluorescent fusion proteins
- Membrane damage in transfected cells
- Cell culture in glass-bottom dishes (Bioptechs)
- Fixation of FDB fibres using 100% ethanol at -20 °C for 5 min
- Application of anti-mouse MG53 rabbit polyclonal antibody at 1:200 dilution
- Application of secondary antibodies coupled with fluorescent probes (goat anti-rabbit Alexa Fluor 488 or Alexa Fluor 546)
- Fluorescence image capture using a BioRad 2100 Radiance laser scanning confocal microscope with a x40 1.3NA oil immersion objective
- Positive control: Not explicitly mentioned.
- Negative control: Not explicitly mentioned.
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