GxTX-594 labeling of surface membranes requires Kv2 protein. GxTX-594 partitioning into the membrane was assessed with fluorescence from nontransfected CHO cells and cells transfected with Kv2-GFP proteins. (A) Fluorescence from live CHO cells transfected with Kv2.1-GFP (top row) or Kv2.2-GFP (bottom row) and labeled with GxTX-594. Airy disk imaging was at a plane above the glass-adhered surface. Cells were incubated with 100 nM GxTX-594 and 5 μg/ml WGA-405 then diluted to 9 nM GxTX-594 and 0.45 μg/ml WGA-405 before imaging. Scale bars, 20 μm. Fluorescence shown was excited at 488 nm (column 1), 594 nm (column 2), or 405 nm (column 3). Scale bars, 20 μm. (B) Fluorescence intensity from Kv2.1-GFP transfected cells with excitation of GxTX-594 at 594 nm versus GFP at 488 nm. Fluorescence from WGA-405 was used as a mask to manually draw ROIs on cells. Each point represents one cell. Cells with obvious GFP fluorescence are green points, cells without are black points. Mean background fluorescence from a region that did not contain cells is indicated by dashed lines. Red line represents a linear fit of cells with obvious GFP fluorescence.