In general, the methodology used to establish cultures of interest from H-2KbtsA58 transgenic mice is to grow cells in whatever conditions favor their division, in the presence of IFN-y at 33°C. Some cell types require a period of growth of at least 24 h in tissue culture before they are cloned, and others can be cloned directly. Cloning can be carried out by limited dilution cloning (as with studies on myoblasts; 78) or can be cloned by infection with a retrovirus encoding a antibiotic resistance gene (as with studies on astrocytes; 66). In the latter case, clonality can be confirmed by Southern blot analysis to ensure that putative clones possess only a single integration site for the antibiotic resistance gene.
Enriched astrocyte cultures were prepared by a modification of the methods of Noble et al. (64). Cortices from perinatal mice were removed and dissected free of meninges, chopped finely, and incubated at 37°C for 30 min in EDTA solution (200 mg/mL solution of EDTA [FDS-grade, Sigma] in Ca2+/Mg2+-free DMEM) containing 8500 U/mL trypsin (Sigma, St. Louis, MO). SBTI-DNase (a solution of 0.52 mg/mL soybean trypsin inhibitor, 0.04 mg/mL bovine pancreatic DNase, and 3 mg/mL BSA fraction V [Sigma]) was added at a ratio of 4 mL for every 7 mL cortical cell suspension and incubated for a further 5 min before being centrifuged at 200g for 5 min. The tissue was resus-pended in DMEM containing 10% fetal calf serum (Imperial), 2 mM glutamine (Gibco-BRL), and 25 mg/mL gentamycin (DMEM-FCS) with 0.4% BSA and dissociated by repeated trituration through 21- and 25-gage needles. The dissociated cells were centrifuged through a cushion of DMEM-FCS containing 4% BSA at 200g for 5 min. The pellet was resuspended in DMEM-FCS and the cells were seeded into flasks coated with 5 mg/mL poly-L-lysine at a density of 107 cells/75-cm2 flask. The cultures were grown for 6 d, and cells on top of the monolayer were removed by shaking overnight at 37°C on a rotary platform (100 rpm). After 24 h, the cells were pulsed with 2 x 10-5 M cytosine arabino-side (AraC) for 4 d. This procedure routinely produced astrocyte cultures of >95% purity as assessed by staining with a polyclonal antiserum against GFAP.
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