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Figure S1. Characterization of microglial cultures. Microglial cells were shaken off from dissociated cell cultures of P2–3 rat cortex and replated. Staining of the replated cells with antibodies against the microglial markers, (A) Iba-1 and (B) lectin, demonstrated that the established cultures predominantly (≥99%) consist of microglial cells. Cell nuclei were visualized by DAPI. Scale bar, 25 μm.

Figure S2. Time-course of SDF-1-dependent activation of Erk1/2 and Akt signalling in primary microglial cells. Cultured primary rat microglia were treated for the indicated time with SDF-1 (100 ng/ml) and subsequently analysed for activation (phosphorylation) of (A) Erk1/2 and (B) Akt by Western blotting and the use of phosphospecific antibodies. Protein loading was controlled by additionally staining blots with antibodies recognizing Erk1/2 and Akt independent of their phosphorylation status. Immunoreactive protein bands were quantified by densitometry and corrected for protein loading. Levels of phosphorylated (p)Erk1/2 and pAkt present in untreated controls were set to 1. Bars represent mean ± SD (n = 3). Levels of both pErk1/2 and pAkt were maximal after 3 min of SDF-1 treatment and returned to control levels after 6 min and 10 min respectively. **P < 0.005; *P < 0.02, treatment vs. untreated control.

Figure S3. Comparative analysis of the chemotactic response of microglial cells to various chemokines and to ATP. The chemotactic response of primary rat microglial cells was tested using a modified Boyden chamber. Cell migration was determined after 4 h. The number of cells that migrated in the absence of any compound was set to 1. Bars represent the mean number of migrated cells ± SD relative to controls (migration index) as determined in three to four independent experiments. The chemokines MCP-1, MIP1α and SDF-1 induced microglial cell migration with similar potencies. Compared with chemokines, ATP more potently stimulated microglial migration. *P < 0.05; **P < 0.001.

Figure S4. Effects of LPS on CXCR4 and CXCR7 cell surface expression. Cultured primary microglia were treated with the indicated concentrations of LPS for 24 h and subsequently analysed for (A) CXCR4 and (B) CXCR7 cell surface expression levels by flow cytometry. Expression levels of CXCR4 and CXCR7 determined in untreated controls were set to 1. Bars represent mean expression levels ± SD as determined in five to six independent experiments. Cell surface expression of both chemokine receptors increased following treatment with LPS at 1 ng/ml, but not following treatment with higher concentrations. *P < 0.001, treatment vs. untreated control.

Figure S5. Time-course of CXCR4 and CXCR7 expression in microglia cells following experimental stroke. Rats were subjected to unilateral permanent MCAO and sacrificed after the indicated times. Brains were removed, cyrosectioned, and double stained with antibodies against Iba-1 (red) and CXCR4 or CXCR7 (both green). On the lesioned (ipsilateral) side, microglia located adjacent to the infarct core showed increased expression of CXCR4 72 h after MCAO whereas increases in CXCR7 expression became first detectable after 96 h (see Figure 5). Expression levels of CXCR4 and CXCR7 remained high for at least up to 240 h following MCAO. Further note that Iba-1-positive microglial cells, present in the contralateral (non-injured) hemisphere, express only extremely low levels of CXCR4, and are virtually devoid of CXCR7. Scale bar, 50 μm.

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