Figure 1.

Cell characteristics and Desmin expression of satellite cells (SC)/myogenic precursor cells (MPC) liberated with trypsin alone or with a combined trypsin, collagenase, and DNase digestion (TCD). a–c Trimmed muscle fragments from SM and LD muscle of 4- to 5-d-old piglets were digested with 0.25% trypsin alone or with a combination of 0.25% trypsin with 0.2% collagenase and 0.01% DNase and enriched with Percoll density gradient centrifugation. (a) Cell yield, viability, and cell size of isolated cells were determined directly after isolation with the countess automated cell counter (Invitrogen, Karlsruhe, Germany) with integrated trypan blue staining. Mean cell yield is illustrated as bars, and individual values are given by circles. Viability and cell size are represented as means ± SD, n = 5 piglets. Digestion with TCD liberated more SC/MPC than digestion with trypsin alone, but the differences between treatments showed only a statistical trend (p = 0.096). Cell viability and cell size were similar for both digestion procedures. For the statistical analysis, a paired t test was performed, and p ≤ 0.05 was considered statistically significant. (*b, c) Cells were immunostained for Desmin and were analyzed with flow cytometry (b) or microscopically (cell nuclei were stained with DAPI) (c). Flow cytometric analyses were performed at various time points between day 3 and day 11 of culture. For both digestion procedures single values from at least five different isolations and the resulting trend lines (T) are presented. The immunofluorescence stainings were conducted at day 7 of culture and results are representative of three individual experiments from three animals. Scale bar represents 50 μm. Desmin expression increased during culture and reached about 60% at days 6–8 in the two differently liberated SC/MPC cultures.