Figure 2

Images and cytometry of stably-expressed GluA1o-γ4 293-F cells. Cells were labelled with (a–c) stable expression of GCaMP6s and (d–f) fluo-4. Pseudocolors represent the intensity with 488 nm excitation (green; GCaMP6s or fluo-4) and 405 nm excitation (blue; Hoechst 33342). (a,d) First column in each group: cells treated with 10 µM CP-465022 to fully inhibit AMPAR-mediated calcium flux. Second column in each group: cells treated with 10 µM glutamate and 10 µM LY-395153. Third column in each group: cells treated with 10 µM ionomycin to fully saturate the indicator in all labelled cells. The first row shows the fluorescence intensity at 488 nm excitation. The second row shows the fluorescence intensity at 405 nm excitation. The third row is an overlay of the first and second rows. Scale bars are 20 µm. (b,e) Vertical projections of overlay images. The narrow side panels at the top and right edges of the main image show vertical projections along the faint overlay lines. The small square image at the top is a magnification of the box marked ‘x, y’ in the main image. The adjacent square images show the z-axis projections of this box. (c,f) Histograms of fluorescence intensity by flow cytometry of cells in suspension: CP-465022 (red), glutamate plus LY-395153 (orange), ionomycin (blue).