Janelia fluor 549

Halo-ligands conjugated to Alexa Fluor 488, Janelia Fluor 549, and Janelia Fluor 646 fluorophores (Promega) were reconstituted to 1 mM in DMSO. Halo-tagged proteins were mixed with fluorophores in a 1:1 concentration ratio, then incubated on ice for 1–2 hours in the dark. After Halo-ligand conjugation reactions were complete, unreacted fluorophores were removed with micro-spin columns loaded with dye removal purification resin (Pierce) at 4 °C. Proteins were then diluted to 2–8 μM in MBOS (Motility Buffer + Oxygen Scavengers: 20 mM MOPS pH 7.4, 5 mM MgCl₂, 0.1 mM EGTA, 50 mM KCl, 15 mM glucose, 50 mM DTT, 20 μg/mL catalase (Sigma C3515), and 100 μg/mL glucose oxidase (Sigma G6125)) + 1–4 mM ATP. Aggregates were removed by ultracentrifugation at 4 °C at 100,111 × g in a TLA100 rotor (Beckman). Fresh Halo-fluorophore conjugated proteins were prepared for each experiment.

Halo fusion proteins were visualized in cells by staining with fluorescent Halo ligand (Promega GA1110). Cells were seeded on polyD lysine-coated coverslips (neuVitro) in full growth media supplemented with 2 μg/mL doxycycline to induce Halo transgene expression for 48 hr. Following a PBS wash, cells were incubated with Halo ligand Janelia Fluor 549 (Promega GA1110) at 25 nM for 30 min at room temperature. Following PBS wash, cells were fixed with 3.7% formaldehyde for 20 min at room temperature. Cell nuclei were stained with 100 ng/mL DAPI (Sigma-Aldrich D9542) for 15 min, and then cells were washed again with PBS. Coverslips were mounted with fluoromount G (SouthernBiotech 0100–01) and imaged at ×60 magnification with a Deltavision Elite widefield fluorescence microscope (GE). DAPI was imaged with 5% transmittance with an exposure of 0.3 s. Halo staining was visualized in the TRITC channel with 10% transmittance and an exposure of 0.2 s. Z stacks were collected of approximately 68 images, 0.2 µm apart for a total thickness of 13.6 µm.

Immunofluorescence assays were performed as described previously (Krtková et al., 2016 (link)), To detect triple hemagglutinin (3HA) tag, anti-HA rat monoclonal antibodies 3F10 (Roche) diluted to 1:125 followed by Alexa 488-conjugated anti-rat antibody (Molecular Probes) diluted to 1:250 were used. To detect HALO tag 0.5 μM Janelia Fluor 549 (Promega) dye or HaloTag^® TMR Ligand (Promega) were used. CWP1 was detected with Alexa 647-conjugated anti-CWP1 antibody (Waterborne, New Orleans, LA, United States).
Fluorescent images were acquired on a DeltaVision Elite microscope using a 100×, 1.4-numerical aperture objective and a PCO Edge sCMOS camera. Deconvolution was performed with SoftWorx (API, Issaquah, WA, United States) and images were analyzed using Fiji, ImageJ (Schindelin et al., 2012 (link)). Pearson Coefficient, Manders Correlation Coefficient and Costes’ automatic thresholding analyses were obtained using the JACoP plugin for ImageJ (Bolte and Cordelières, 2006 (link)). 3D viewing and manual scoring of cells were performed using Imaris (Bitplane, version 8.9). Figures were assembled using either Adobe Photoshop or Adobe Illustrator. A minimum of 120 cells were imaged for each cell line and timepoint post induction of encystation which corresponded to between 15 and 20 cells at each of our defined stages.