2. Harvest the culture at OD 600 0.3–0.4 by pouring 21.85 mL of
the yeast culture in a 50 ml plastic tube and adding 3.15 mL of
32% (w/v) paraformaldehyde. Mix gently and incubate at RT
for 45 min with constant shaking (see Note 6).
3. Centrifuge the cells at 2400 Â g for 3 min at 4
C and wash
three times with 10 mL of ice-cold buffer B.
4. Resuspend cells in 1 mL of ice-cold buffer B and transfer them
to a 1.5 mL tube.
5. Centrifuge cells at 2400 Â g for 3 min at 4
C, remove all
buffer B, and resuspend cells in 500 μL of spheroplasting buffer
containing the Lyticase (use 25 U of enzyme per mL X OD 600
of cells, i.e., 21.85 mL of cultures at OD 600
~0.4 ¼ 21.85 Â 0.4 ¼ 8.74; thus, add 8.74 μL of Lyticase
per sample).
6. Incubate the cells in a water bath at 30
C for 7–8 min, inverting gently and frequently (see Note 7).
7. After 5 min of Lyticase treatment take 5 μL sample on a slide,
cover it with a coverslip, and observe the cells with phasecontrast illumination. After Lyticase treatment, cells should
appear opaque.
8. Take samples every 2 min and stop the Lyticase treatment when
50% of the cells in the sample are opaque (Fig. 1).
~50% digested spheroplasts
digested
non-digested
Fig. 1 Yeast cells treated for 7–8 min with Lyticase. Digested cells are imaged
using phase-contrast illumination. Upon cell wall digestion, cells appear opaque.
The treatment is stopped when 50–60% of the cells in the sample are opaque.
Scale bar ¼ 20 μm
Detection of mRNA and Proteins in Single Cells by smFISH-IF
57