Biomedical Engineering Reference
In-Depth Information
2,105 cm
−1
(CD
2
stretching vibrations [
27
,
28
]). Both, CH
2
and
CD
2
stretching vibrations are detected in epi(E)-CARS mode
using suitable emission fi lters (650/210, 770SP) and a non-
descanned detector (NDD) [
29
,
30
]. The appropriate wave
number is set via the user interfaced of the SP5 microscope control
software.
Fiji is an ImageJ-based public domain image processing software
(
http://fi ji.sc
;
Wayne Rasband, National Institute of Mental
Health, Bethesda, Maryland, USA). Leica LAS AF ver. 2.6.3 (Leica
Microsystems, Inc.) is used for microscope control and basic image
processing.
2.7 Image
Processing Software
3
Methods
3.1 Cell Cultivation
Fluorescence labeling effi ciency (BP 493/503, LD540, and
specifi cally Nile Red) and GFP expression levels are strongly depen-
dent on cell growth phase and cell viability. Thus, great care has to
be taken to maintain reproducible growth conditions. A more
homogeneous labeling in a cell population can be achieved by two
to three cycles of pre-cultivation, 8-12 h each (
see
Note 1
).
1. Cultivate yeast cells in culture fl asks containing 5 ml YPD for
12 h at 30 °C on a rotary shaker (180 rpm).
2. Inoculate 100 ml fresh YPD in a 500 ml fl ask with 100
3.1.1 Stationary
Phase Cells
l of
the preculture and cultivate cells for 72 h, to stationary phase.
μ
1. Cultivate yeast cells in culture fl asks containing 5 ml YPD
medium for 12 h at 30 °C on a rotary shaker (180 rpm).
2. Inoculate 100 ml fresh YPD medium in a 500 ml fl ask with
100
3.1.2 Logarithmically
Growing Cells
l of the preculture and cultivate cells for 72 h, to station-
ary phase.
3. Inoculate 2-10 ml fresh YPD medium with 1/100 volume of
stationary phase culture and cultivate for 4-8 h at 30 °C on a
rotary shaker (180 rpm).
μ
3.2 Separation
of Quiescent Yeast
Cells by Density
Gradient
Centrifugation
This protocol is based on the method described by Allen et al. for
the isolation of quiescent cells from stationary phase cultures [
31
].
The procedure proofed to be extremely valuable for microscopic
investigation of cells due to the quantitative elimination of necrotic
or apoptotic cells and stationary phase cells with large vacuoles.
Thus, vital staining and GFP expression patterns turn out to be
very homogeneous in the cell population (
see
Note 2
).
1. Add 9 ml RediGrad™ and 1 ml sterile 1.5 M NaCl to 38 ml
high-speed centrifuge tubes (e.g., COREX™ glass tubes).
