Ron Lab


 

Staining tunicamycin treated C. elegans with DCF

3/27/03

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 (20μl) 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 (25-50μl) aliquots at –20C.
  • M9
  • 30% sucrose in H20 filter sterilized and prechilled to 4C.
  • Ice cold 0.1M NaCl

Protocol

  • Worms are treated with tunicamycin by making a stock of 5μg/ml TM in 500μl M9 and spreading it onto a 60mm plate of growing worms overnight.
  • 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.
  • The worms are then resuspended in 500μl DCHF-DA (2mm, in M9) and incubated on a rotator/rocker for 1hour wrapped in foil.
  • The worms are centrifuged at 3000rpm and washed in 1ml M9.
  • 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.
  • The worms are rapidly resuspended in 1ml ice cold 30% sucrose that is then layered over with 250μl ice cold 0.1N NaCl and centrifuged in at 5000rpm in a 4C table top centrifuge for 5 min.
  • 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.
  • The worms are then immobilized and embedded in aggarose for photography.
  • 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