Use of a 96-well plate reader to evaluate proliferation of equine satellite cell clones in vitro

Abstract

We have adapted a methylene blue staining assay to measure proliferation of equine satellite cell clones in a 96-well format. This technique allows rapid and accurate measurement of proliferating satellite cells which is a considerable enhancement over manual counting methods. Methylene blue is incorporated into the nuclei and intracellular matrix of satellite cells and then released into the aqueous phase. Absorbance of stained cells is read at 620 nm and correlates with increasing numbers of cells (range tested from 1 × 103 to 4 × 104). This method was used to determine the response of equine satellite cells to FGF, both human recombinant and bovine, and to IGF-1. This format is very efficient in measuring and comparing the proliferation of equine satellite cells. However, fusion of cells to form multinucleated myotubes cannot be assayed using this method because it lacks the sensitivity and specificity to differentiate multinucleated from mononucleated cells, and to detect expression of myogenic proteins. The assay could be accurately applied from 0 to 144 hours, before significant fusion and differentiation takes place. Using this assay will reduce analysis time to quantitate the proliferation response of equine satellite cells to different growth factors.