Figure 7.

Amacrine cell survival in vitro. (A) Amacrine cells were purified and plated at different densities in serum-free medium. Cells were counted at 1, 2, or 3 DIV after adding calcein-AM and the nuclear dye DAPI. Survival was calculated as the percentage of cells that were calcein⁺ of the total number of calcein⁺ and DAPI⁺ cells. (N = 2; n > 150 for each condition. Error bars: SEM). (B, C) Acutely purified P8 amacrine cells were plated at low density in growth medium containing peptide trophic factors and forskolin, with and without pharmacologic inhibitors (see below). Survival was quantified at 3 DIV with calcein/PI (N > 3; n > 150 for each condition; ***P < 0.001, one-way ANOVA with the Tukey post hoc test; **P < 0.01, *P < 0.05, paired t-test. Error bars: SEM). (D, E) Amacrine cells were purified and incubated for 2 hours in growth medium with or without inhibitors as labeled, after which they were centrifuged and processed for protein extraction. Example Westerns are shown; graphs are the average of at least two experiments, normalized to NB Sato (*P < 0.05, **P < 0.005; unpaired t-test. Error bars: SEM). B, BDNF; C, CNTF; F, forskolin; I, insulin; NB, Neurobasal+penicillin/streptomycin; NB Sato, Neurobasal+Sato stock+pyruvate+penicillin/streptomycin; GM, growth medium; U0, U0126; K, K252a; LY, LY294002; A, AG490; D, DMSO; FS, full Sato.