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In experiment with PrP mutants, cells were grown in coverslips and loaded with Fluo-4 AM (3 µM) for 30 min at room temperature.

When cytoplasmic MT effectors Dyn1p or Kar9p were deleted in S172A and S172E mutants, cells were viable but presented increased ploidy.

To create gene deletion mutants, cells were transformed with linearized plasmids or PCR products bearing a selection marker (URA3, LEU2 or KanMX genes) flanked by sequences complementary to the genes to be deleted.

In the activated macrophages, the reduction of CFU counts in the mutant was much more pronounced, at 6-days of post infection, 0.5 logs of the mutants cells were recovered (Fig. 5).

For melan-d1 complementation assays measuring the function of myosin mutants, cells were seeded in a 24-well plate and allowed to reattach prior to infection with adenovirus.

To isolate can1 R mutants, cells were plated on SD-Arg containing 60 mg/liter canavanine and resistant colonies were picked.

For example, the mutant cells were significantly longer and presented multiple aberrant septa.

cbpB mutant cells were also more sensitive to osmotic stress than wild-type cells.

The arrested unsynchronized mutant cells were mostly dumbbell shaped (data not shown), consistent with an S phase-arrested morphology.

In contrast, under both non-limiting and limiting nutrient conditions, C. reinhardtii mutant cells were cleared equally (Van Donk 1997).

At the end of the cultivation period, the supernatant was discarded after the mutant cells were pelleted by centrifugation.