Microscopic and Spectroscopic Techniques to Investigate Lipid Droplet Formation and Turnover in Yeast - Membrane Trafficking - page 291

Bacto™ agar (BD, Inc., USA).
12-well microtiter plates (Greiner, Inc.).

For preparation of yeast media, yeast extract (1 % w/v) and
peptone (2 % w/v) are weighed in an appropriate fl ask and fi lled up
with distilled water to 9/10 of the fi nal volume. Autoclave at
121 °C for 20 min; add 1/10 volume of fi lter-sterilized 10× glu-
cose solution (2 % w/v fi nal concentration) to the media, mix well,
and dispense into sterile fl asks or glass tubes. For solid media plates,
2 % Bacto™ agar is added to the solution prior to autoclaving.

BODIPY 493/503 (Invitrogen, Inc.). 1 mg/ml stock solution in
DMSO.
LD540 ([ 21 ] see below). 1 mg/ml stock solution in DMSO.
Nile Red (Sigma-Aldrich, Inc). 1 mg/ml stock solution in DMSO.
Oleic acid- D 34 (Sigma-Aldrich, Inc.). 1 % (w/v) stock solution in
10 % Tergitol.
Palmitic acid (Sigma-Aldrich, Inc.). 1 % (w/v) stock solution in
10 % Tergitol.
Tergitol NP-40S 70 % stock solution (Sigma, Inc.).
Tergitol working solution 10 % (aqua dest. diluted stock
solution).
Tris base (Merck, Inc.).
D -Sorbitol (Merck, Inc.).
NaCl (Merck, Inc.).
Agarose electrophoresis grade (Invitrogen, Inc.).
RediGrad™ (Amersham Biosciences, Inc.).

2.3 Dyes
and Reagents

2.4 Cell Preparation

COREX™ high-speed glass centrifugation tubes (38 ml).
Thermomixer (Eppendorf Comfort, Eppendorf, Inc.).
50 ml plastic tubes (Greiner, Inc.).

2.5 Fluorescence
Microscopy Hardware
and Imaging Software

Microscope slides 76 × 26 mm (Roth, Inc.).
Coverslips 50 × 24 mm #1, 0.17 mm (Menzel, Inc.).

Leica Microsystems SP5 confocal microscope with differential
interference contrast DIC (transmission imaging), spectral detec-
tion, and sequential scanning feature. HCX PL APO 63×/1.4 OIL
CS oil-immersion objective. HCX PL APO 63×/1.2 W CORR
water-immersion objective. Leica LAS AF ver. 2.6.3 (Leica
Microsystems, Inc.) microscope control software.

2.6 CARS
Microscopy Hardware
and Imaging Software

The CARS (coherent anti-Stokes Raman scattering) system is
based on a Leica SP5 confocal microscope (Leica Microsystems,
Inc.). The CARS signal is generated using a picosecond laser source
with integrated optical parametric oscillator ( pico Emerald; APE,
Germany; HighQ Laser, Austria). For the detection of neutral lip-
ids and lipid droplets, the laser is tuned to 2,845 cm −1 , which
enables the imaging of CH 2 symmetric stretching vibrations.
To detect deuterium-labeled fatty acids, the laser is tuned to