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ijs12197-sup-0001-si.doc1953K

Fig. S1. Immunostaining of primary brain endothelial cells (BECs) with rabbit polyclonal anti-von Willebrand factor antibody (a), rat astrocytes with rabbit polyclonal anti-glial fibrillary acidic protein (GFAP) antibody (b), pericytes with rabbit monoclonal anti-platelet-derived growth factor receptor beta (PDGFRb) and mouse monoclonal anti-neuronal-glial 2 (NG2) proteoglycan antibodies (c) and microglia with rabbit polyclonal anti-CD11b antibody (d).

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Fig. S2. Cell necrosis and late apoptosis of astrocytes measured by Annexin PE/7-AAD staining.

Primary astrocytes were exposed to six-hour control conditions (a) or six-hour OGD (b). Cells were detached and an aliquot of ≈500 000 cells were stained with Annexin V – Phycoerythrin (PE) and 7-Amino-actinomycin (7-AAD) according to manufacturer's instruction (BD Pharmigen), giving a total volume of 0·5 ml. Quantification of stained cells was done in Beckman Cytmoics FC 500 flow cytometer. 7-AAD fluorescence was plotted versus Annexin PE fluorescence. Cells located in the B4 quadrant were early, apoptotic, 7-AAD-negative/Annexin PE-positive. Cells in the B3 quadrant were viable, 7-AAD-negative/Annexin PE-negative. Cells in the upper quadrants B1 (7-AAD-positive/annexin PE-negative) and B2 (7AAD-positive/annexin PE-positive) were necrotic and late apoptotic, respectively. A representative panel from each group is shown.

ijs12197-sup-0001-si.doc1953K

Table S1. Effect of time on concentration of angiogenic factors in the cell culture medium during OGD and in controls – one-way ANOVA.

ijs12197-sup-0001-si.doc1953K

Table S2. Cell necrosis and late apoptosis of astrocytes measured by Annexin PE/7-AAD staining and a comparison of these data with percentage of dead cells estimated by LDH assay.

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