January 2013

Discussion

Conclusion

Future Aspects

Introduction

Methodology

Cellular stains

All Nuclei

r

DNAHoechst 33342

Human Nuclei

r

Nuclear envelopePrimary [1:2000]: Monoclonal rabbit-anti-human LaminSecondary [1:400]: Alexa Fluor 555 conjugated goat-anti-rabbit

Cytoplasm

r

Green Fluorescent ProteinEx: 487; Em: 509

Myosin Heavy Chain

r

Primary [1:500]: Rabbit-anti-myosinSecondary (10+10 ul/cm2): Zenon Alexa Fluor 647 mouse-anti-rabbit IgG

Cell line development

ASC-GFP

C2C12

C2C12-GFP

C2C12-LifeAct-RFP

Mechanical Stimulation

Uniaxial cyclic tensile strain

Flexcell setup

r

6 well plates or the new design?

Results

Measures

Directionality

Degree of differentiation

Morphology

Size

No of syncytial nuclei

Murine nuclei

Human nuclei

Presence of Myosin

Expectations

Control

Low degree of fusion (<5%)

Unlikely >1 human nuclei per myotube

Stimulated cells

Alignment, perpendicular to direction of strain

Higher involvement in myotube formation

Outcomes