Myoblast isolation and fluorescence microscope visualization
For this purpose, two different approaches were tested: 1) single cell
C1 Fluidigm platform (Fluidigm, South San Francisco, CA) and 2) sorting
cytometry. The first approach, single cell C1 Fluidigm platform, was
especially suitable for our purpose because isolated cells can be
visualized through microscopy and after, RNA extraction and cDNA
synthesis can be made in an automated system that works with thermal
cycling and pneumatic controls, minimizing the variation between
conditions and expending only small amounts of reagents in every plate.
To make the analysis with the C1 Fluidigm single cell platform,
myoblasts were resuspended in C1 suspension reagent and loaded into the
C1 IFC chip, that contains 96 single cell capture sites. After this
process, myoblasts were captured in single and isolated chambers in the
IFC and then observed by fluorescent microscopy at 40X to count foci
number. Unfortunately, we failed to detect foci fluorescent signal (see
results section), so we decided to do this process through our second
approach, sorting cytometry. For this second approach, cells were sorted
using FACSAria II flow cytometer (BD Biosciences, San Jose, CA) into
96-Well Optical-Bottom Plates. For each participant, we sorted a total
of 120 FISH stained myoblasts distributed in two plates. The cytometer
could not detect the fluorescent signal of the FISH staining (probably
because RNA foci fluorescence signal is small and because of their
nuclear localization). Therefore, sorting was only used to isolate
individual myoblasts. RNA foci signal was observed by fluorescent
microscopy. In order to spend the least time possible, since these same
cells were later used for RNA analysis, and because there were two
plates to analyze per participant, two independent observers
simultaneously visualized one plate each, in similar fluorescence
microscopes. They had the same criteria for annotating RNA foci number
in every single myoblast by naked eye visualization.