Imaging the mitotic spindle by spinning disk microscopy in tobacco suspension cultured cells

Plants are valuable systems for analyzing the acentriolar mitotic spindle. We have developed methods for imaging the mitotic spindle in living tobacco (Nicotiana tabacum) suspension culture cells expressing GFP-α-tubulin. The methods allow the spindle to be observed in living cells at high spatial and temporal resolution and rely on water immersion objectives, spinning disk optics, and high-sensitivity cameras. Here, we describe these methods and provide step-by-step protocols for certain key steps. We also describe a method for application and removal of inhibitors.