This protocol allows researchers to observe how C-elgans respond to acetylcholine receptor agonist Levamisole. Altered Levamisole sensitivity can suggest defects in signaling at their neuromuscular junction or muscle function. A major advantage is that the vigorous swimming of C-elgans in the Levamisole solution allows researchers to quantitative the time dependent paralysis of hundreds of worms in just one hour.
Counting the number of worms in each well every five minutes can be overwhelming. Number of wells assayed, or time points can be adjusted if necessary. To begin prepare nematode growth media by combining three grams of sodium chloride, 2.5 grams of pep tone, and 17 grams of agar with one liter of deionized water in a flask with a stir bar.
After autoclaving, put the media on a hot plate set to 70 degree Celsius and stir at a moderate speed for one hour. Add one milliliter of five milligrams per milliliter cholesterol dropwise to prevent precipitation. One milliliter of one molar calcium chloride, one milliliter of one molar magnesium sulfate, and 25 milliliters of one molar pH 6.0 potassium phosphate buffer to the media.
Transfer two milliliters of nematode growth media into each well of a 24 well plate with a sterile serological pipette. Allowed the plates to dry on the bench top for two days before seating with bacteria. Pick a single op 50 colony into B broth and set the culture shaking at 37 degrees Celsius overnight.
Using a sterile pipette, drop 30 microliters of OP50 suspension onto the agar in the middle of each well. let the plates sit at room temperature for at least two days after seating to allow a bacterial lawn to form. Grow wild type unc-63 Lev10 and unc-49 C-elgan to adulthood on six centimeter plates, preparing at least eight plates per strain.
Prepare bleaching solution under the hood on the day of synchronization. Mix 10 milliliters of bleach, 2.5 milliliters of sodium hydroxide, and 37.5 milliliters of deionized water in a 50 milliliter chronicle tube. Using a plastic transfer pipette wash gravid adult worms from at least four plates with M9 buffer and transfer them into a 15 milliliter chronicle tube.
Spin that 716 G for one minute at room temperature and then remove the super natant using a transfer pipette. Add 10 milliliters of the bleaching solution. Gently shake the tube for four minutes until most, but not all of the worm carcasses have dissolved.
Spin at 716 G for one minute. Pour off the bleach solution in one motion. As long as the tube is not shaken at this point the eggs will stick to the side of the tube at 15 milliliters of M9 buffer and invert.
Spin at 716 G for one minute and pour off the M9 buffer in one smooth motion. Repeat this washing with buffer solution three times. After the final wash, add 10 milliliters of fresh M9 and place on a rotator overnight at 15 degrees Celsius to isolate a synchronized population of starved first larval stage animals.
Print out to 24 well plate and assign strains to randomized places. Approximately 24 hours after the bleach prep, spin down hatched starved L1 worms at 716 G for one minute at room temperature. Remove approximately nine milliliters of M9 buffer with a plastic transfer pipette, and then gently mix the starved first larval stage worms in the remaining M9 buffer.
Immediately pipette three microliters of the worms and M9 buffer onto a microscope slide and determine the number of L1's. The desired number is 20 to 30 L1's in three microliters. Pipette three microliters of L1's into each well according to the pre-made plate map, let the worms grow to adulthood for three days at 20 degrees Celsius.
Print the blank data sheet which will be used to record the number of worms moving in each well every five minutes for one hour. Check the worms in the 24 well plates. Using a marker, make an X on the plate lid over any wells that have contamination, have starved, or have too many worms, which will make counting difficult.
Make 0.4 millimolar Levamisole solution by adding 200 microliters of 100 millimolar levamisole stock to 50 milliliters of M9.Start a timer and then using a transfer pipette add one milliliter of 0.4 millimolar of levamisole to the first two wells, such that the animals are freely swimming. Continue to add Levamisole to the adjacent wells, staggering the time according to the number of wells to be assayed. At five minutes, start manually counting only the number of moving worms in each well, beginning with the first well, and record that number on the data sheet.
Continue to count the number of moving worms in each well every five minutes for one hour. At the end of the assay or when time permits, record the total number of worms in each well. Obtain the plate, map and record which strain corresponds to each well.
Enter the data into a spreadsheet starting with the total number of worms in each well, and organizing by genotype. Combining data from the wells, determine the number of worms moving at each time point. Use this to calculate the number that paralyze at each time point.
Make a data table such that the left hand column indicates time, and subsequent columns contain the data for each strain. For each animal that is paralyzed within the first five minutes, create a row and enter a one for five minutes. Repeat this for each time point.
For all animals that do not paralyze by the end of the assay, enter a zero for 60 minutes. Use these data to create a survival curve in the specific statistical software used here to visually display the time-dependent paralysis of the population. Data for all strains assayed must be entered into the same data table, leaving spaces as necessary.
Mutations in subunits of the Levamisole acetylcholine receptor, as well as in genes required for clustering of post synaptic Levamisole sensitive acetylcholine receptors, cause resistance to Levamisole induced paralysis as observed in the unc-63 and Lev-10 mutants respectively. Loss of the GABA-gated ion channel unc-49 caused Levamisole hypersensitivity due to disruption of the proper balance of cholinergic and GABAergic signaling. Proper preparation of 24 well plates is essential.
If bacterial lawns are not dry before spotting L1's the Levamisole solution can become cloudy during the assay, which inhibits observation. By modifying the 24 well plates, this protocol can be performed with RNAi knocked down animals. This allows researchers to study C-elgan's genes, for which mutants are not available.
