Uplansapo 20x objective

Wild type S. pombe L972 cells were grown in liquid EMM medium containing 0.5% glucose at 30°C overnight shaking at 200 rpm, diluted and re-grown to mid-log phase the next day. Cells were harvested by centrifugation, washed twice with medium or buffer containing 1.2 M sorbitol and applied to a 4-chamber glass-bottom dish (Greiner BIO-ONE) coated with concanavalin-A. For energy depletion experiments, cells were energy-depleted as described above prior to loading to the dish. Unbound cells were washed off with EMM medium or phosphate buffers containing 1.2 M sorbitol. Bound cells were covered with 400 µl of phosphate buffer of different pH containing 1.2 M sorbitol and either 2 mM DNP (pH experiment) or 20 mM 2-DG and 10 µM antimycin A (energy depletion experiment). Cells were imaged for five frames before addition of 40 µl cell wall digesting enzymes (final concentrations: 0.5 mg/ml Zymolyase 100T, 2.5 mg/ml lysing enzymes from Trichoderma harzianum). Spheroplasting and rounding up of cells was followed by time-lapse bright-field microscopy with a DeltaVision (Applied Precision) microscope (Olympus IX70 stand, Olympus UPlanSApo 20x objective, CoolSnap HQ2 camera).