Deltavision microscopy system

HeLa cells transfected with Mog1 siRNA or mCerulean-Mog1 were fixed using PTEM buffer (60 mM PIPES, pH6.8, 10 mM EGTA, 2 mM MgCl₂, 0.2% Triton X-100) supplemented with 3.7% paraformaldehyde. After blocking with 1% bovine serum albumin (Sigma-Aldrich) in PBST (PBS with 0.05% Tween-20) buffer for 45 min at room temperature, the fixed cells were incubated with primary antibodies in a humidified chamber for 1 h followed by secondary antibodies for 1 h at room temperature. The DNA was stained with DAPI (Sigma-Aldrich). Images were acquired by DeltaVision SoftWoRx software (Applied Precision) and processed by deconvolution and z-stack projection.
For live cell imaging, HeLa cells were cultured in glass-bottom culture dishes (MatTek) and maintained in CO₂-independent media (Gibco) supplemented with 10% (v/v) FBS and 2 mM glutamine (Ding et al., 2010 (link)). During imaging, the dishes were placed in a sealed chamber at 37°C. Images of living cells were taken with a DeltaVision microscopy system (Applied Precision Inc.). Image processing was performed with SoftWoRx (Applied Precision Inc.). To trace chromosomes in mitosis, frames were collected at 3–5 min intervals. Images were prepared for publication using Adobe Photoshop software.

For immunofluorescence analysis, HFF cells were grown on coverslips and inoculated with T. gondii parasites in the absence and presence of ATc (at 1 mg/ml) for 48–50 h. To investigate the distribution of actin in extracellular parasites, growth in ATc was extended to 68–72 h, and freshly released tachyzoites were harvested. Cells were fixed with 4% (wt/vol) paraformaldehyde and 0.0075% glutaraldehyde (Electron Microscopy Sciences, Hatfield, PA) in phosphate-buffered saline (PBS) for 10 min before permeabilization with 1% (vol/vol) Triton X-100 in PBS for 10 min, followed by blocking in 3% (wt/vol) bovine serum albumin (Sigma-Aldrich, St. Louis, MO) in PBS for 1 h. Primary antibodies were diluted in blocking solution and used in the following concentrations: rabbit anti-HA clone C29F4 (Cell Signaling Technology, Danvers, MA), 1:4000; mouse anti-cMyc clone 9E10 (Sigma-Aldrich), 1:2000; and rabbit anti-Act_239-253 (Angrisano et al., 2012 (link)), 1:4000. Secondary antibodies used were Alexa Fluor 488 goat anti-mouse and Alexa Fluor 594 goat anti-rabbit (Life Technologies) in 1:4000 dilutions.
Fluorescence microscopy was performed using a DeltaVision Microscopy System (Applied Precision, Seattle, WA) and an Olympus UPlanSApo 60×/numerical aperture (NA) 1.4 or 100×/1.4 NA oil objective.

MDA-MB-231 cells grown on glass-bottom culture dishes (MatTek) were starved overnight and then stimulated with 20% serum. Images were taken with a DeltaVision microscopy system (Applied Precision Inc.) at a rate of 1 frame/10 min. Cell movement was tracked by Image-Pro Plus 6.0. Total migration velocity (υ_T) is total track distance divided by total time, and directional migration velocity (υ_D) is the direct distance from starting point to ending point divided by total time.