Snap surface 549

MEFS, COS-7, or CHO cells were seeded sparsely and grown for 2 d on 35-mm cell culture dishes fitted with a glass coverslip at the bottom. Cells were transfected with the different SNAP-tagged CD44 plasmids 16–18 h prior to the experiment using FuGENE 6 Transfection Reagent. Labeling was done with SNAP tag-specific photo-stable fluorescent probes, SNAP Alexa 546, SNAP-surface 549 (λex/λem: 560/575 nm, purchased from New England Biolabs, Ipswich, MA), or JF646-SNAP ligand (λex/λem: 646/664 nm) by incubating for 10 min at 37°C using a dilution of 30 nM (for single particle experiments) and 50–100 nM (for cartography experiments) with 10% serum containing F12 medium and then washed extensively with glucose-M1 buffer (150 mM NaCl, 5 mM KCl, 1 mM CaCl₂, 1 mM MgCl₂, 20 mM HEPES, pH 7.3; supplemented with d-glucose at 2 mg/ml) to get rid of free dyes. The dyes were chosen to ensure they are spectrally different from GFP with minimum bleed-through. Dual color labeling was done with JF549-cpSNAP ligand (λex/λem: 549/571 nm) and JF646-SNAP ligand (λex/λem: 646/664 nm) fluorophores by incubating for 10 min at 37°C with F12 serum medium at mixed concentrations of 50 and 150 nM for the respective dyes. Singly or dually labeled cells were subsequently washed and imaged at 37°C in the presence of HEPES buffer containing 2 mg/ml glucose.