Fluorescent Labeling of Live Cells

HeLa cells (ATCC) and U2OS cells (ATCC) were cultured in Dulbecco’s modified eagle medium (DMEM; Life Technologies) supplemented with 10% v/v fetal bovine serum (FBS; Life Technologies), 1 mM GlutaMax (Life Technologies), and 1 mM sodium pyruvate (Sigma) and maintained at 37 °C in a humidified 5% v/v CO₂ environment. These cell lines undergo regular mycoplasma testing by the Janelia Cell Culture Facility. Cells were transfected with HaloTag–H2B, HaloTag–tubulin, SnapTag–TetR, or SnapTag–H2B using an Amaxa Nucleofector (Lonza). Before the imaging experiments, transfected cells were transferred onto a No.1 coverslip (Warner Instruments) that was cleaned by Piranha solution (3:1 v/v mixture of concentrated H₂SO₄ and 30% v/v hydrogen peroxide). To label live cells with the HaloTag or SnapTag ligands, compounds 9, 10, 27, 28, 29, 30, or 31 were added to the growth medium and the samples incubated for 15 min. Labeling concentrations were typically 100–500 nM for confocal, wide-field, and dSTORM experiments and 5–50 nM for single-molecule tracking experiments. Cells were then washed briefly with PBS (1×) and then incubated in DMEM–FBS for an additional 15 min. Before imaging, the cells were washed briefly with PBS (3×) and placed in fresh DMEM–FBS for imaging. All washes were omitted in the “no wash” experiments. For nuclear staining, cells were incubated in PBS for 5 min (2×), and then incubated in PBS containing 5 μM DRAQ5 (Cell Signaling) for 5 min, followed by brief wash with PBS (1×). During all imaging experiments, cells were maintained at 37 °C in a humidified 5% CO₂ v/v environment supplied by a live-cell incubator (TOKAI HIT).