Biomedical Engineering Reference
In-Depth Information
2. Centrifuge cells at 1,000 ×
g
for 1 min in a tabletop centrifuge;
aspirate off supernatant.
3. Resuspend cells in 1 ml sterile 50 mM Tris-HCl, pH 7.5
4. Add 1
l of the Nile Red stock solution to the cell suspension
(fi nal concentration, 1
μ
g/ml). Vortex briefl y.
5. Incubate cells for 20 min at RT. Do not close the lid of the
reaction tube during staining.
6. Centrifuge cells at 1,000 ×
g
for 2 min using a tabletop centri-
fuge. Aspirate off supernatant and resuspend cells in the
remaining liquid.
7. Mount 1
μ
l of dense cell suspension on a standard microscope
slide for short-term analysis or on a microscope slide covered
with agar (
see
Subheading
3.3
), for long-term observation.
μ
For labeling logarithmically growing or stationary phase cells, and in
combination with GFP detection, cells are preferentially fi xed with
formaldehyde prior to Nile Red staining (
see
Notes 9
and
10
).
3.5.4 Nile Red Staining
of Logarithmically Growing
Cells
1. Transfer 1 ml of yeast culture to a 1.5 ml reaction tube.
2. Centrifuge cells at 1,000 ×
g
for 1 min in a tabletop centrifuge.
Aspirate off supernatant.
3. Resuspend cells in 500
μ
l sterile 1 M sorbitol. Incubate for
60 s at RT.
4. Add 27
l of a 37 % formaldehyde solution (v/v) to the sample
(fi nal concentration 2 %, v/v). Incubate cells for 1-5 min at
RT (
see
Note 10
). Vortex briefl y every 30 s.
5. Centrifuge cells for 2 min at 1,000 ×
g
in a tabletop
centrifuge.
6. Wash cells three times with 500
μ
μ
l sterile 50 mM Tris-HCl
pH 7.5.
7. Add 0.2
l of the Nile Red stock solution to the cell suspension
(fi nal concentration, 0.4
μ
g/ml); vortex briefl y.
8. Stain cells for 10 min at room temperature.
9. Centrifuge cells at 1,000 ×
g
for 2 min using a tabletop centri-
fuge. Aspirate off supernatant and resuspend cells in the
remaining liquid.
10. Mount 1
μ
l of dense cell suspension on a standard microscope
slide for short-term analysis or on a microscope slide covered
with agarose (
see
Subheading
3.3
), for long-term observation.
μ
3.6 Fatty Acid
Treatment of Yeast
Cells for CARS
Microscopy
1. Cultivate yeast cells to the desired growth stage (
see
Subheading
3.1
).
2. Add 100
l of the deuterium-labeled oleic acid stock solution
(fi nal concentration, 0.05 %) and 100
μ
l of the unlabelled pal-
mitic acid stock solution (fi nal concentration, 0.05 %) to 2 ml
μ
