Fig. 7

Stimulation of the P2X7R causes robust permeabilization of human microglia to exclusively cationic dyes. a Stimulation of the P2X7R with BzATP (300 μM) induced the uptake of the cationic dye YO-PRO-1 in human microglia. Representative fluorescence images of cells incubated for 15 min in the presence of 300 μM ATP +/− the antagonists A804598 (20 μM) or A438079 (50 μM). Each antagonist was pre-incubated for 30 min. Scale bar: 20 μm. b Quantitative comparison of YO-PRO-1 uptake by microglia. Cells were incubated for 15 min with YO-PRO-1 and 300 μM BzATP in the presence of various inhibitors: BX430 (10 μM), A438079 (50 μM), and A804598 (20 μM), n = 4 separate experiments; there was significant inhibition by P2X7R antagonists (p < 0.0001; ANOVA). c Time course of YO-PRO-1 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 +/− A438079 (50 μM). d, e Activation of the P2X7R with BzATP (300 μM) does not induce the uptake of the anionic dyes Lucifer yellow (Lucifer yellow, 0.5 mM) or carboxyfluorescein (CF, 0.5 mM) in human microglia. d Example fluorescence images of cells incubated in the presence of 300 μM ATP with the anionic dyes Lucifer yellow or CF. e Quantitative comparison of anionic dye uptake by microglia. Cells were incubated for 15 min with Lucifer yellow or CF in the presence of BzATP (300 μM). There is no significant difference between dye uptake in control and treated cells (n = 4 separate experiments). f Unlike human microglia, J774A.1 mouse macrophages display strong uptake of anionic dyes. Mouse macrophages were incubated for 15 min with Lucifer yellow (0.5 mM) or CF (0.5 mM) in the presence of ATP (5 mM), n = 3 separate experiments; there was significant difference of dye RFU after ATP treatment (p < 0.0001; unpaired t test)