Figure 7

Müller cell expression of chemotactic cytokines following microglia co-culture. (A) Quantitative RT-PCR comparing mRNA levels of chemotactic cytokines in Müller cells cultured alone (control, white bars), or co-cultured with unactivated (gray bars) or activated microglia (black bars). The mRNA levels of CCL2 and CCL3 were significantly elevated in Müller cell co-cultured with activated microglia, compared with those cultured alone or cultured with unactivated microglia. Expression levels of CX3CL1 were unchanged between the three groups. Representative gel images for genes whose expression were significantly changed following co-culture are shown (right). (B) Protein levels of chemokines CCL2 and CCL3 in Müller cell-conditioned media were measured with ELISA. Relatively elevated levels of these chemokines were found in conditioned media from Müller cells co-cultured with activated microglia. (C) The ability of Müller cell-conditioned media to induce the chemotaxis of microglia was assayed using a modified Boydon chamber. Conditioned media from Müller cells exposed to activated microglia exerted the largest chemoattractive effect on microglia (black bar), followed by media from Müller cells co-cultured with unactivated microglia (gray bar), relative to media from Müller cells that were cultured alone (white bar). CCL2 (100 nM in DMEM medium) was used as a positive control for microglial chemotaxis. (* indicates p < 0.05 for all comparisons, one-way ANOVA with Tukey-Kramer multiple comparison test, n = 6-8 replicates from two independent experiments.)).