Methods

3.1. Subcellular 1. The murine hippocampal cell line HT22 has been used exten-

Fractionation sively to examine oxidative stress-induced cell death (12,

13). HT22 cells are routinely passaged when approaching confluence with trypsin/EDTA and seeded in 100-mm culture dishes at a density of 1 x 10 5 cells/100 mm dish (see Note 4). For oxidant exposure studies, cells should be seeded at a density of 2.5 x 10 5 cells per 100-mm dish and the following day exposed to oxidants. To ensure that enough protein is obtained for each subcellular fraction, three 100-mm dishes (approx 1 x 106 cells) should be used for each experimental treatment and pooled. As a positive control, cells should be treated with either 1 mM diamide (a disulfide-promoting agent) or 5 mM H2O2 for 5 min (see

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