For immunofluorescence, HeLa cells were plated onto a sterile 24-well plate (Corning Glass Works) for transfection or drug treatment and fixed with 3.7% formaldehyde in PBS for 10 min at 37°C. In order to facilitate the staining of the antibody into the cells, the treated and fixed cells were permeated with PBS containing 0.1% Triton X-100 for 5 min. After blocking with PBS with 0.05% Tween-20 buffer containing 1% bovine serum albumin (Sigma) for 45 min at room temperature, the fixed cells were incubated with primary antibodies in a humidified chamber for 1 h at room temperature or overnight at 4°C, followed by secondary antibodies for 1 h at 37°C. The DNA was stained with 4′,6-diamidino-2-phenylindole from Sigma. Images were captured by DeltaVision softWoRx software (Applied Precision) and processed by deconvolution and z-stack projection.
For time-lapse imaging, HeLa cells were cultured in glass-bottom culture dishes (MatTek) and maintained in CO2-independent media (Gibco) supplemented with 10% FBS and 2 mM glutamine (Mo et al., 2016 (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.
For time-lapse imaging, HeLa cells were cultured in glass-bottom culture dishes (MatTek) and maintained in CO2-independent media (Gibco) supplemented with 10% FBS and 2 mM glutamine (Mo et al., 2016 (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.
