Methodological considerations for heat shock of the nematode Caenorhabditis elegans

Abstract

Stress response pathways share commonalities across many species, including humans, making heat shock experiments valuable tools for many biologists. The study of stress response in Caenorhabditis elegans has provided great insight into many complex pathways and diseases. Nevertheless, the heat shock/heat stress field does not have consensus as to the timing, temperature, or duration of the exposure and protocols differ extensively between laboratories. The lack of cohesiveness makes it difficult to compare results between groups or to know where to start when preparing your own protocol. We present a discussion of some of the major hurdles to reproducibility in heat shock experiments as well as detailed protocols for heat shock and hormesis experiments.

Keywords: Heat shock; Heat stress; Hormesis.

Figure 1

Changes in agar temperature with time. Agar plates moved from a maintenance temperature (e.g. 20°C) to a 37°C incubator do not come immediately up to the temperature of the incubator. The most dramatic increase in temperature occurs within the first 5 minutes. Time -1 = room temperature immediately before being placed into the incubator, time 0 = immediately after being placed onto the pre-warmed gel pad and covered with other warm gel pads as well as possible without disturbing the thermocouple probes. Temperatures were recorded using a thermocouple attached to a digital thermometer (Omega Engineering model HH506RA).

Figure 2

Life stage differences in heat shock responsiveness. (A) Survival was compared by scoring percent (%) survival of N2 wild-type worms at each larval stage. Worm growth plates were removed from 37°C as indicated. Three or four plates/ time point were counted for each developmental stage. L1 and L2 survival was assessed 2 days post-HS (Since HS causes a developmental delay, allowing worms to fully recover increased our confidence that only worms that will ultimately survive to adulthood and produce offspring were counted). L3 and L4 survival was assessed 12 -14 hours post-HS. There are no significant differences from 1.0 to 2.5 hrs when L1 worms are exposed to 37°C. Differences in survival following heat shock are significant beginning at 1.5 hrs of exposure to 37°C (compare L1 to L3 and L4, P=0.008 & 0.014, respectively, one-way ANOVA w/ Bonferroni correction). (B) Differences in survival of adult N2, wild-type worms when comparing worms exposed to 1.5 hours at 37°C at Day 1, 2, 3, or 4 of adulthood [71, 95, 119, 143 hours post egg-lay, respectively). Two plates were prepared as described in (A) for each age tested. Survival of day 1 worms is significantly different from day 2 and day 4 worms (P=0.02 and 0.02, respectively). Comparing day 1 to day 3 worms (P=0.054), they are not significantly different, but this is likely due to the small sample size in this experiment. Note: 1.5 hours was chosen for this experiment to ensure no plate had 0% survival.

Figure 3

Incubator set up for heat shock experiments. The image below shows our efforts to get as much warm surface area as possible into the incubator. Shown is a Forma Scientific water-jacketed, stacked incubator, model 3327. The top shelf was chosen for the experimental space because, without the fan running, it was the shelf that most frequently stayed at the temperature set on the incubator. The middle shelf tended to be lower than desired and the bottom shelf lower still. Although this was less than a 1°C difference from top to bottom, that can be enough to affect the outcome of an experiment. The bottom shelf has a desktop file organizer filled with gelatinous freezer packs and a small battery-operated fan. The gel pad on the second shelf is used on top of the experimental plates during heat shock.

Figure 4

Differences in survival following heat shock when eggs are laid directly onto plates compared with bleach-prepped eggs. Laid-egg plates were prepared as described in Section 3.2.1. Bleach-prepped eggs were prepared using a Wormbook protocol for decontaminating worm stocks [67]. The worms described as short bleach-prep were in lysis buffer approximately 10.5 minutes, only long enough for the worms to lyse, spin down, and be rinsed. The long bleach-prep worms were left in lysis buffer a total of approximately 14.5 minutes. Five plates were prepared for each condition and placed into a 37°C incubator for 1.5 hours as described in Section 3.2.1. 1.5 hours was chosen for this experiment instead of two to make sure no plates had 0% survival. The number of worms/plate ranged from 63 to 180 with an average of 105 worms/plate. Mean survival on the laid-egg plates was 21% and is significantly different from the short bleach-prepped plates, which had a mean survival of 45% and the long bleach-prepped plates, which had a mean survival of 52% (P=0.0359 and 0.0039, respectively, one-way ANOVA w/ Tukey's multiple comparison test).