Fluorescent K+ Sensor Bead Generation

K⁺ sensor beads were generated by first covalently linking human IgG (Sigma) and fatty acid-free bovine serum albumin (Sigma) to 12.5 mg of carboxylated, 3 μm silica beads (Kisker Biotech) as previously described [2 (link), 68 (link)]. Beads were then washed once with coupling buffer (0.1 M sodium borate, pH 8.0), and twice with PBS. 20 mg of EDC (Sigma) and 25 μg of Asante Potassium Green 4, TMA+ salt (TEFLabs) were added to the beads in 1 ml of PBS, and the mixture incubated with agitation for 2 hours at room temperature, protected from light. APG4 beads were washed three times with PBS, before resuspension in 1 ml of coupling buffer containing 25 μg of Alexa Fluor 594-SE (Invitrogen), and incubation with agitation for an additional 1 hour at room temperature, protected from light. APG4/AF594 beads were washed twice with coupling buffer, and once with PBS, before final resuspension in 500 μl of PBS.