Additional file 5: of A central role for P2X7 receptors in human microglia

Figure S4. P2X7R dye uptake is independent of cell lysis and selective for cations below a certain threshold of molecular weight. (a) Time course of ethidium uptake shows the average s.e.m. of fluorescence intensity over time measured from individual cells within a single field of view after application of 300 μM BzATP (red trace) compared to unstimulated microglia (black trace). (b) Quantification of ethidium uptake by microglia reveals significant stimulation of dye uptake by BzATP (300 μM) for 25 separate experiments. Significance determined using an unpaired t test. (c) Quantitative comparison of YOYO-1 uptake by microglia. Cells were incubated for 15 min with YOYO-1 and 300 μM BzATP. There was no significant uptake of the dye, n = 4 separate experiments. As a positive control, microglia were lysed with 0.5% Triton X-100 and displayed robust uptake of YOYO-1. (d) Human microglia were treated with BzATP (300 μM), ATP (5 mM), or nigericin (20 μM) for 30 min at 37 °C. The negative control was obtained from untreated microglia, and the positive control was from lysed microglia. After treatment, cell supernatants were collected and LDH release was quantified. Percent cytotoxicity is the amount of LDH release normalized to the positive control; there was no significant difference between the negative control and BzATP, ATP, or nigericin-treated microglia (n = 3 separate experiments). (EPS 5922 kb