Methylcellulose

Serial dilutions of MAb were incubated with 100 FFU of CHIKV for one hour at 37°C. MAb-virus complexes were added to cells in 96-well plates. After 90 minutes, cells were overlaid with 1% (w/v) methylcellulose in Modified Eagle Media (MEM) supplemented with 4% FBS. Plates were harvested 18 to 24 hours later, and fixed with 1% PFA in PBS. The plates were incubated sequentially with 500 ng/ml of ch-CHK-9 and horseradish peroxidase (HRP)-conjugated goat anti-human IgG in PBS supplemented with 0.1% saponin and 0.1% BSA. CHIKV-infected foci were visualized using TrueBlue peroxidase substrate (KPL) and quantitated on an ImmunoSpot 5.0.37 macroanalyzer (Cellular Technologies Ltd). Non-linear regression analysis was performed, and EC50 values were calculated after comparison to wells infected with CHIKV in the absence of antibody.

Resveratrol (RES) was purchased from Supelco (MO, United States) and prepared freshly in an appropriate vehicle (VEH) consisting of 1% carboxyl methyl cellulose (CMC, Sigma-Aldrich, United States). SEB was obtained from Toxin Technology Inc. (FL, United States) and aliquoted in sterile phosphates buffer saline (PBS) at a stock concentration of 2 μg/μL prior to storage at −20°C until being used.

Double-cycled MT seeds were prepared by combining TRITC-labeled (49%), biotinylated (18%), and unlabeled tubulin (33%; Cytoskeleton) reconstituted in MRB80 (80 mM K-Pipes, 1 mM EGTA, 4 mM MgCl₂; pH 6.80 with KOH) to a final concentration of 20 µM with 1 mM GMPCPP (Jena Bioscience) on ice. The mixture was incubated at 35°C for 30 min to allow MTs to polymerize. Seeds were pelleted by centrifugation in an airfuge (Beckman coulter) at 20 psi for 5 min, resuspended in MRB80, and depolymerized on ice for 25 min. The tubulin was then repolymerized upon the addition of fresh GMPCPP by incubating at 35°C for 30 min. These seeds were pelleted by centrifugation in an airfuge at 20 psi for 5 min, resuspended and diluted sixfold in MRB80 supplemented with 10% [vol/vol] glycerol, aliquoted, flash-frozen in liquid nitrogen, and stored at −80°C until use.
To prepare the chambers, a clean glass coverslip was plasma-treated and fixed to a clean glass slide using strips of double-sided tape to create two parallel chambers of ∼10 µl. The surface was blocked and functionalized by incubating with a mix of 95% PLL-g-PEG and 5% PLL-g-PEG-biotin (0.1 mg/ml in 10 mM Hepes, pH 7.40; SuSoS) for 10 min. After washing with MRB80 supplemented with 40% [vol/vol] glycerol (MRB80-gly40), NeutrAvidin was introduced and incubated for 10 min. After washing, 50-fold diluted GMPCPP seeds were introduced and incubated for 5 min before washing once more and then incubating with Κ-casein for >3 min.
All reaction mixtures (MT mix, expansion mix, rigor mix, washout mix) were prepared at double the volume for the paired compacted/expanded lattice samples and split into two equal parts prior to the addition of DMSO (compacted control) or 20 µM Taxol (expanded). Reagents were added to MRB80-gly40 such that the effective glycerol concentration in the MT mix was 20% and in the other mixes was ∼27%. All mixes contained 0.1% [wt/vol] methylcellulose, 0.5 mg/ml K-casein, 50 mM glucose, 0.2 mg/ml catalase, 0.5 mg/ml glucose oxidase, and 10 mM DTT. The MT mix additionally contained 1 mM GTP, 10.8 µM porcine tubulin (Cytoskeleton), and 0.6 µM TRITC-labeled porcine tubulin (Cytoskeleton). The expansion mix additionally contained 50 mM KCl and 20 µM Taxol (or the equivalent dilution of DMSO). The rigor mix additionally contained 50 mM KCl, 20 µM Taxol (or the equivalent dilution of DMSO), 2 mM ATP, and 15.2 pM StableMARK. The washout mixture additionally contained 50 mM KCl, 20 µM Taxol (or the equivalent dilution of DMSO), and 2 mM ATP. After preparation, these mixtures were spun in an airfuge at 20 psi for 5 min, transferred to clean tubes, and kept on ice until use.
Samples were then moved to the TIRF microscope equipped with a stage-top incubator to maintain them at a constant temperature of 30°C. MTs were grown by flowing in two chamber volumes (ChV) of the MT mix and letting it incubate for 15 min. Subsequently, the chambers were flushed with five ChV MRB80-gly40. Next, the lattices were (mock) expanded by adding two ChV expansion mix (or DMSO equivalent) and incubating for 10 min. Next, two ChV rigor mix was added and incubated for 90 s. Finally, four ChV washout mix was added before imaging. For imaging, the following sequence was used: 2 × Taxol, 4 × DMSO, 4 × Taxol, 4 × DMSO, and either 2 × Taxol or 4 × Taxol and 2 × DMSO (8 or 10 images/condition/assay), and images were taken at similar heights within the channels.