This
protocol was used in our recent publication (Harding, et al., 2003, An integrated stress response
regulates amino acid metabolism and resistance to oxidative stress. Mol
Cell; 11:619)
Reagents:
•DCHF-DA
(Dichlorodihydrofluoresceine diacetate, Molecular probes D-399) is dissolved in
DMSO to 50mM and stored at –80C in small (20ul) aliquots wrapped in
foil. Do not freeze-thaw more than
twice.
•Tm (tunicamycin (Calbiochem or Sigma) is dissolved to 5mg/ml
in DMSO and stored in small (25-50ul) aliquots at –20C.
•M9
•30%
sucrose in H20 filter sterilized and prechilled to 4C.
•Ice
cold 0.1M NaCl
Protocol:
1.
Worms are treated with tunicamycin by making a stock of 5ug/ml TM in 500ul M9
and spreading it onto a 60mm plate of growing worms overnight.
2. The
next morning the worms are washed off the plate and collected by centrifugation
at 3000RPM in M9 in a 15ml tube, transferred to 1.5ml tubes and washed twice in
1ml M9.
3. The
worms are then resuspended in 500ul DCHF-DA (2mm, in M9) and incubated on a
rotator/rocker for 1hour wrapped in foil.
4. The
worms are centrifuged at 3000rpm and washed in 1ml M9.
5. The
following steps are very important because it removes aggregated DCF, bacteria,
and dead worms that can give high background. However, steps 6 & 7 must be preformed rapidly to
prevent osmotic shock induced toxicity to the worms.
6. The worms are rapidly resuspended in 1ml ice cold 30% sucrose
that is then layered over with 250ul ice cold 0.1N NaCl and centrifuged in at
5000rpm in a 4C table top centrifuge for 5 min.
7. The
worms are on the interphase and are collected by transfering the upper phase to
a new tube containing1ml M9. The
worms are pelleted at 3000rpm and washed again 2-4times in 1 ml M9. It is preferable to spin the worms in
swinging bucket rotor.
8. The
worms are then immobilized and embedded in aggarose for photography.
9.
Because DCF is oxidized by UV light, it is best to photograph worms randomly
from one side of the slide to the other.
For example starting on the left side, the first worm is identified and
photographed with transmitted visible light. Then the fluorescent light is
opened and a fluorescent micrograph is taken. The slide is then move over to an
area past where the light has increased the background by oxidation and the
fluorescent light is closed. Then
the next worm is identified and photographed sequentially with transmitted and
then fluorescent light using the same settings as the first worm. This process is repeated across the width
of the stage. After
comparing the unadjusted micrographs of several worms from untreated treated
and treated worms it became apparent that the TM treated worms were much
brighter than their untreated counterparts. Ero1 RNAi
blockage of the DCF signal was also readily apparent when pictures were
taken in this unbiased way. If the
UV light is exposed to fields of worms for extended amounts of time, all the
worms will eventually become very bright.
Likewise, it is not a good idea to go back over areas that you have
already taken pictures, because the worms that were directly in the light
previously will be brighter than the ones that have never been exposed. A microscope equipped with quantitative
fluorometric capabilities might also be useful in these experiments.
Marcie Calfon, PhD & Heather Harding, PhD