Ix2 slp

CMs were isolated enzymatically from LV as described before^{35 (link)}. After isolation, CMs were pre-plated on a T-75 culture flask in Medium 199 (ThermoFisher Scientific) with 1% of penicillin/streptomycin (ThermoFisher Scientific) and incubated for 4 hours so that the non-CM cell population could attach to the culture flask. The supernatant with the CMs was collected from the culture flask after 4 hours and CMs were plated onto laminin (10 µg/mL; Sigma) coated coverslips, in 6-well plates for 3 hours. For direct 2-D co-cultures, Fbs or MyoFbs were added to the 6-well plate, in a ratio of 2.5 (Fb/MyoFb):1 (CM). The previously obtained Fbs/MyoFbs were cultured in CM medium with 2.5% fetal bovine serum for 24 hours before they were seeded onto the CM culture. To set up 2-D indirect co-culture systems, we used a 0.4 µm Thincert™ (Greiner Bio-One) preventing direct contact of Fbs with CMs but allowing the Fb secretome to diffuse to CMs. After 24 and 48 hours of co-culture, we quantified viability of CMs using light microscopy (Olympus IX2-SLP with MotiCam 3 camera), by counting the density of rod-shaped striated CMs and expressing the count as percentage of the density at day 0. We further evaluated structural remodeling in the live cells by counting the fraction of CMs with lateral or distal spreading.

The procedures were as described previously (Lee et al. 2012 (link)). Briefly, the heart rate of embryos was first counted for 1 min under a dissection microscope at 27 °C, then the embryos were anaesthetized with 0.06 % MS222 (pH 7.25, Sigma-Aldrich), and their images recorded under a microscope (IX2-SLP, Olympus, Tokyo, Japan) from 1 to 3 days post fertilization (dpf) and at hatch. The eye length, width, and pigmentation density (eye density), the distance between the eyes (eye distance; representing the head growth), the width of the optic tectum (midbrain width), and the hatchling body length were analyzed from the images with ImageJ image processing and analysis software (http://rsbweb.nih.gov/ij/). All images were obtained and analyzed under identical conditions without any alteration.

A total of 100,000 cells per well in 0.35% agarose growth medium were seeded in 6-well plates over a base of 0.7% agarose medium. The day after seeding, 3 mL of complete growth medium was added to each well. The media was replaced twice a week until the end of the experiment, at which point the colonies had undergone sufficient growth. Then, photos were taken of each well with microscope (#IX2-SLP, Olympus) with an installed camera (#U-CMAD3, Olympus), and colony number and size were determined.