Isolation of Primary Muscle Cells from mdx Mice

Primary muscle cells were isolated from 3-wk-old mdx mice (C57BL/10ScSn mdx/mdx, The Jackson Laboratory) using a technique previously described (Rando and Blau 1994; Qu et al. 1998). The isolated cells were then suspended in the growth medium (DME supplemented with 10% FBS, 10% horse serum, 0.5% chick embryo extract, and 2% penicillin/streptomycin). The cells were then preplated in collagen-coated flasks (Rando and Blau 1994; Qu et al. 1998). After ∼1 h, the supernatant was withdrawn from the flask and replated in a fresh collagen-coated flask. The cells that adhered rapidly within this 1-h incubation were mostly fibroblasts (Rando and Blau 1994; Qu et al. 1998; Qu and Huard 2000a,Qu and Huard 2000b). The serial replating of the supernatant was repeated when 30–40% of the cells had adhered to each flask. After ∼5–6 serial platings, the culture was enriched with small, round cells (pp6; Qu et al. 1998; Qu and Huard 2000a,Qu and Huard 2000b).