Figure 3
Spontaneous global organization increases myotube lengths. (a) Representative composite image (n = 4) of F-actin in C2C12 myotubes, spanning a full well in a standard 12-well plate. Composite images were composed of 252 frames, and the OrientationJ ImageJ plug-in was used to false-color the image according to the local angle of orientation of F-actin filaments. Top: C2C12 culture on 10% w/v gelatin—10 U/mL TG hydrogels. Bottom: C2C12 culture on 2.5% w/v gelatin—10 U/mL TG hydrogels. Close-up areas: 1cm2, scale bars 5 mm (b) Length of myotubes developed on a range of hydrogel substrates: gelatin: 2.5–10% w/v, TG: 0.6–10 U/ml. Middle bar represents median length, while the upper and lower bound of the box respectively represents the upper and lower quartile. Lines indicate largest and lowest value within 1.5 times the interquartile range. For each gel composition, four separate cultures were used to evaluate at least 100 random myotubes. Statistical significance: *P < 0.05, ***P < 0.001 (c) Distributions of myotube lengths for 2.5, 5 and 10% (w/v) gelatin hydrogels, cross-linked using 10 U/mL TG. Evaluation performed at day 9 after initiation of differentiation.
