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- Dissolve the FM dye to a stock concentration of 100 μM in 1×
PBS, pH 7.4. Protect from light and place on ice. - Add 20 μl of the stock to each sample (fi nal concentration of
FM dye is 1 μM). - Cover tubes with aluminum foil to protect dye from light.
- Label vacuoles by further incubating cells with dye at 30 °C,
225 rpm for 1 h ( see Note 12 ). - After incubation with dye, immediately place the cells on ice.
- Add 270 μL of 37 % (v/v) formaldehyde solution to the sam-
ple and further incubate on ice for 15 min. - Transfer mixture to microcentrifuge tube and collect the cells
by centrifugation 5,600 × g for 2 min. - Discard supernatant and resuspend the cells in 500 μL of 1× PBS.
- Collect the cells via centrifugation as in step 10.
- Resuspend the cells in 200 μL of 1× PBS. The cells may be
stored at 4 °C for 24 h. - Spot 15–20 μL of fi xed cells on microscope slide.
- Place coverslip. Protect sample from light.
- View and take pictures of the cells on the microscope (Fig. 2 ).
- Grow cells in 5 mL of YPD media to an OD 660 between 0.4
and 0.5. - Transfer 1 mL of the culture to a microcentrifuge and collect
cells via centrifugation at 1,400 × g for 5 min. - Resuspend the cells in 1 mL of cold 70 % (v/v) ethanol.
3.7 DNA Staining
Fig. 2 Vacuolar morphology in yeast. S. cerevisiae BY4741 cells were stained
with the lipophilic styryl dye, FM 4–64 for 1 h. Images of stained cells were cap-
tured using AxioVision 4.8 software on a Zeiss Axio Observer Z1 microscope
(63× magnifi cation). The above representation is a magnifi cation of the area of
interest depicting variable morphology and number of vacuolesAmit Shrestha et al.http://www.ebook3000.com