These methods can be used to characterize stress responses in C.elegans and to determine whether genetic or pharmacological perturbations affect the activation of protective cellular pathways. These methods allow rapid and high-throughput testing of hundreds of perturbations such as gene knockdown both on the molecular and physiological levels. To ensure the assay is performed properly, include positive and negative controls.
Synchronize healthy, well-fed animals to the proper stage and use fresh plates for the experiment. Visualization of micromanipulation of the worms with a pick can help new users understand how the worms can be lined up and assessed for viability. Demonstrating the procedures will be Phil Frankino, Holly Gildea, and Melissa Metcalf, graduate students in our laboratory.
To use animals expressing GFP under the promoter of the heat shock protein four for activation of the endoplasmic reticulum unfolded protein response, grow synchronized reporter animals at 20 degrees Celsius until the L4 stage and wash the worms off the plate using M9 medium and transfer into a tube. After collecting the worms by centrifugation, replace the M9 with 25 nanograms per milliliter of the drug of interest in M9 or dimethyl sulfoxide in M9 in the control animal tubes. Then place the worms on a rotating platform for three to four hours at 20 degrees Celsius.
At the end of the incubation, centrifuge the worms to settle before replacing the treatment solution with 15 milliliters of M9 alone. After two washes, transfer the animals to NGM plates or NGM RNA interference plates as appropriate and allow the worms to recover overnight at 20 degrees Celsius. When the worms have recovered, add five to 10 microliters of 100 millimolar sodium azide onto a standard NGM plate without bacteria and place a plate of worms under a dissecting microscope.
Transfer 10 to 20 animals from the plate into the spot of sodium azide. The animals should seize movement shortly after landing in the salt solution. Once the sodium azide has evaporated, line up the animals in the desired imaging setup with the anterior and posterior sides in the same orientation for all of the animals and immediately place the plate of animals under a stereo microscope.
Launch the stereo microscope imaging software and under the acquisitions tab, click open projects and folder to open a new project. Right-click and select rename to rename the folder. Position the worm sample under the microscope objective and use the Brightfield setting to locate the correct focal point of the worms to minimize fluorescent bleaching.
The center of the sample will be the point at which the line of eggs is clearly visible and not fuzzy. Set the exposure time to ensure that pixels are not saturated and also to ensure that the signal is above the detection limit. After setting the exposure time, zoom, focus, and Brightfield condensers to the appropriate settings, click the capture image button to acquire an image.
For imaging using a compound wide-field microscope, place the baseline control treatment plate onto the compound wide-field microscope stage and use the touch pad to launch the control program. Create a new album and filename and position the plate under the objective lens. Use the baseline control and positive control plates to set the exposure time and fluorescence intensity so that the signal is visible but not saturated.
Then save Brightfield and GFP FITC images. To quantify the induction of the reporter by large particle flow cytometry, first turn on the cytometer lasers and open the cytometer software. Click start to turn on the lasers in the software window and click run to initiate the laser in the argon laser control popup window.
When the laser reaches around 12 milliwatts and the 488 light source level goes up to around 12, click done and check the four pressure values. If the values look similar to those observed in the original setup, check the pressure OK box. Next, to make sure there are no air bubbles or debris blocking the flow of sheath and sample through the flow cell, click clean several times.
Then switch off sort and on sheath to restart the flow. To check the flow rate, collect the sheath for 60 seconds. Collect the sheath into a 15 milliliter tube for 60 seconds.
The flow rate should be nine to 10 milliliters per minute. To run the samples, adjust the laser photomultiplier power to a level high enough that the signal is above the detection limit but not higher than the saturation limit. Perform size gating to exclude bubbles, debris, eggs, and other unwanted small particles and to include only the animals of interest such as adults.
When the screening parameters have been set, add the prepared worms to a cup and click acquire. Watch to make sure that all of the liquid is not taken up into the machine as this will cause the flow cytometer to take in air and create bubbles within the detector. When the sample is low and/or enough animals have been collected, click stop.
To store gated data based only on the size constraints, click setup, data storage, only gated, and store gated to save the data. Then rinse the collection cup with deionized water and remove the wash by vacuum three times before repeating the analysis with the next sample. To measure the mitochondrial and oxidative stress sensitivity, add 50 to 75 microliters of Paraquat in M9 to eight to 10 wells per condition of a flat bottom 96-well plate and transfer eight to 10 worms per condition to each well of Paraquat.
Every two hours, tap the plate gently to cause the live animals to thrash or bend to allow scoring of the number of dead animals per well. To measure the temperature sensitivity of the animals, incubate the worms for three to four days at 20 degrees Celsius before plating 10 to 15 animals per plate per condition for a total of four to six plates. Then place the plates into a 34 or 37 degree Celsius incubator and score the worm survival every two hours as just demonstrated.
The hsp-4 reporter has minimal basal impression in the absence of stress but exhibits a robust GFP expression when the animals are exposed to endoplasmic reticulum stress using the drug Tunicamycin. These differences can also be quantified using a large particle flow cytometer. Moreover, the induction of the transgene under endoplasmic reticulum stress can be completely supressed by knockdown of XBP-1 via RNA interference.
Similar assays can be performed to measure mitochondrial stress, oxidative stress, and heat stress. Whole animal physiological responses to stress can also be measured using several survival assays. For example, exposure to Tunicamycin is used to measure endoplasmic reticulum stress sensitivity.
The knockdown of the XBP-1 gene results in a significant increase in sensitivity to Tunicamycin. Moreover, exposure to the chemical agent Paraquat is used to measure oxidative and mitochondrial stress sensitivity. Knockdown of the DAF-2 gene results in a significant increase in resistance to Paraquat.
Thermotolerance can be measured by determining the survival of animals at elevated temperatures and can be plotted as a survival curve. These assays should be performed at least four to six times and all of the replicates should be plotted against each other as thermotolerance demonstrates an incredibly high variability as compared to other stress assays. When imaging animals, it is critical to set the parameters such that there is no over or under saturation of signal and to ensure that the same parameters are used throughout the experiment.
The imaging protocols described here are amenable for large scale screening including genome-wide screens and are great for both exploratory and confirmatory studies which can then be followed up by the physiological assays described.
