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. 2005 Mar 16;25(11):2952–2964. doi: 10.1523/JNEUROSCI.4456-04.2005

Figure 5.

Figure 5.

TNFα requires microglia to induce neurotoxicity. Cerebellar neuronal cultures were exposed directly to TNFα (0.1 or 0.25 ng/ml) alone (A) or together with control MGCM (B), nonconditioned medium (C), microglia in coculture (D), DCGIV-MGCM (E), or l-AP-4-MGCM (F). Neuronal apoptosis was assessed with Hoechst 33342 staining after 24 h. TNFα was only neurotoxic in the presence of MGCM or microglia in coculture. Data (mean ± SEM) are shown as the percentage of apoptotic neurons per field above respective control (untreated neurons; A, C, D), control MGCM (B), DCGIV-MGCM (E), or l-AP-4-MGCM (F). Significance values are shown compared with respective control: *p < 0.05; **p < 0.005; ***p < 0.0005; p > 0.05 is nonsignificant (ns). G, Caspase-3 activity (CaspaTag; green) and PI staining (red) were assessed in untreated neuronal cultures (i) and those exposed for 24 h to control MGCM (ii), DCGIV-MGCM (iii), 0.25 ng/ml TNFα alone (iv), 0.25 ng/ml TNFα with control MGCM (v), or NAAG-MGCM (vi). Cells were incubated with CaspaTag and PI for 30 min, and staining was viewed using a fluorescence microscope. Where CaspaTag staining and PI staining overlap, cells appear yellow. Gvii, The number of caspase-3- or PI-positive cells were counted and expressed as a percentage of the total cell number per field in at least three fields per coverslip from at least three separate coverslips. Data are expressed as mean ± SEM. Significance values are shown compared with respective CaspaTag or PI controls in CONT MGCM: *p < 0.05; **p < 0.005; ***p < 0.0005; p > 0.05 is nonsignificant (ns).