Apr 07, 2025
Finding NHR mutants with higher sensitivity to nematocide drugs
- John Bergqvist1,
- Julia Riedl1
- 1MRC Laboratory of Medical Sciences, Imperial College London
- Behavioural Genomics
Protocol Citation: John Bergqvist, Julia Riedl 2025. Finding NHR mutants with higher sensitivity to nematocide drugs. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1drkpvr2/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 03, 2025
Last Modified: April 07, 2025
Protocol Integer ID: 123693
Keywords: C. elegans, Nuclear hormone receptors, Nematocidal Drugs, nhr mutants with higher sensitivity, finding nhr mutant, mutants to known nematocidial drug, known nematocidial drug, mutant, nhr, nuclear hormone receptor, higher sensitivity, drugs this protocol, sensitivity, killed worm
Abstract
This protocol explores the sensitivity of C. elegans Nuclear Hormone Receptor (NHR) mutants to known nematocidial drugs using pixel variation as the data output for paralysed/killed worms.
Materials
For a screen of 24 NHR mutants and N2 control (25 strains):
- 50x 100 mm plates
- 25x 60 mm plates
- 75x 15 mL falcon tubes
- 4x 50 mL
- 15x 1.5 mL Eppendorf
- 14x 2 mL Eppendorf
- 25x Round, flat-bottom 96-well NUNCLON plates
- 1x V-bottom, deep-well 96-well plate
- 2x 500 mL Erlenmeyer flasks
- 25x plastic sterile loops
- Chlorpromazine hydrochloride (https://www.sigmaaldrich.com/GB/en/product/mm/215921?srsltid=AfmBOooT3uIvWf6HEaFCowDpdNMnnV32CZQHvH9ssnHeV9YONdGViBX5)
- M9
- S-medium
- DMSO
- Sterile water
- MilliQ water
- 16% Paraformaldehyde
Troubleshooting
Day -5.
Making adult worm population
Pick 20 worms onto a OP50-seeded 100 mm plate with NGM and incubate the worms at 25°C for ~24h.
Making PFA-killed OP50
Make an OP50 O/N culture by pipetting 20 µL of maintenance OP50 culture (OD = 1.0) into 1 L LB broth. Leave shaking at 200 rpm and 37°C O/N.
Day -4.
Making adult worm population
Remove the adults from the plate by picking. Incubate plates at 20°C until Day -1.
Making PFA-killed OP50
Make PFA-treated OP50 (OD = 2.0) out of the OP50 O/N culture using the protocol linked below.
Final resuspension of the pellets should be done with S-medium.
Make Stock 1 concentrations of Aldicarb, Levamisole and Chlorpromazine.
Stock 1 - Aldicarb (24 mM):
Weigh 10 mg in a 5 mL Eppendorf tube. Add 2190 µL of sterile water. Vortex extensively to dissolve.
Stock 1 - Levamisole (50 mM):
Weigh 10 mg in a 2 mL Eppendorf tube. Add 831 µL of sterile water. Vortex extensively to dissolve.
Stock 1 - Chlorpromazine (20 mM):
Weigh 10 mg in a 2 mL Eppendorf tube. Add 831 µL of sterile water. Vortex extensively to dissolve.
Make 1 mL Stock 2 concentrations of Aldicarb, Levamisole and Chlorpromazine in 1.5 mL Eppendorf tubes.
Stock 2 - Aldicarb (4.8 mM)
Add 200 µL of Stock 1 Aldicarb (24 mM) to 800 µL sterile water. Vortex.
Stock 2 - Levamisole (1.2 mM)
Add 24 µL of Stock 1 Levamisole (50 mM) to 976 µL sterile water. Vortex.
Stock 2 - Chlorpromazine (1.2 mM)
Add 60 µL of Stock 1 Chlorpromazine (20 mM) to 940 µL sterile water. Vortex.
Store Stock 1 and Stock 2 vials in -20°C.
Day -1.
Estimate the number of eggs on the plates in the morning.
- If there is a high number of eggs on the plate of adult worms, proceed from Step 11
- If there is not enough number of eggs on the plate of adult worms, proceed from Step 9
Wash adult worms off the 100 mm plate and transfer to a 15 mL Falcon tube. Wash once in M9.
Drop the adult worms onto a OP50-seeded 100 mm plates with NGM and incubate at 25°C for 4h.
Wash adults off the plates. Loosen the eggs from the plate using a plastic sterile loop and M9 and transfer to a 15 mL falcon tube. Wash twice in M9 and transfer the eggs to the 60 mm plate with M9.
Leave the 60 mm plate with eggs and M9 in the 20°C incubator O/N to synchronise.
Day 0.
Make Stock 1 concentrations of Ivermectin (always made fresh on the day of the experiment).
Stock 1 - Ivermectin (12 mM):
Weigh 10 mg in a 2 mL Eppendorf tube. Add 952 µL of DMSO. Vortex extensively to dissolve.
Prepare the drug-plate in a deep-well 96-well plate.
N2 concentration determination15 steps
These steps describe the steps done for finding the concentration range that should be applied for the whole screen
Plate layout of the final concentrations in 96 well plates to achive through the below steps (not drug-plate):
Make the working concentrations for Ivermectin in individual 1.5 mL Eppendorf tubes for each 12 wells with different concentrations according to the table below (colour-coded according to the concentrations in the wells and which concentration to use to make a new, lower concentration).
Stock 2 Ivermectin (96 µM):
Add 4 µL of Stock 1 Ivermectin (12 mM) to 496 µL DMSO. Vortex.
Adding Ivermectin to drug-plate:
- In row C, add 3 µL of the Ivermectin concentrations into their respective wells according to the table above.
- Add 97 µL of sterile water to each of the wells in row C. Mix thoroughly by pipetting up and down ~30 times.
Adding Aldicarb to drug-plate:
- In row A, add the volumes of sterile water according to the table below first (colour-coded according to the concentrations in the wells and which concentration to use to make a new, lower concentration).
- Next, add Aldicarb according to the table below and mix thoroughly by pipetting up and down ~30 times.
Adding Levamisole to drug-plate:
- In row B, add the volumes of sterile water according to the table below first (colour-coded according to the concentrations in the wells and which concentration to use to make a new, lower concentration).
- Next, add Levamisole according to the table below and mix thoroughly by pipetting up and down ~30 times.
Adding Chlorpromazine to drug-plate:
- In row D, add the volumes of sterile water according to the table below first (colour-coded according to the concentrations in the wells and which concentration to use to make a new, lower concentration).
- Next, add Chlorpromazine according to the table below and mix thoroughly by pipetting up and down ~30 times.
Adding solvent negative controls to drug-plate:
- In row E, add 100 µL of sterile water to each of the wells.
- In row F, add 3 µL of DMSO to each of the wells. Next, add 97 µL of sterile water and mix thouroughly by pipetting up and down ~30 times.
Using the ViaFlow, add 10 µL of the drugs onto 7 flat and round Nunclon 96-well plates.
Using the ViaFill with the small tubing, prime the tubing. Then dispense 30 µL of the PFA-killed OP50 (OD=2.0) to all plates. Wash the tubing with MilliQ water.
Spin down the now synchronised L1s at Program 1 and wash with fresh M9 once.
Adjust the volume of M9 to acquire ~20 worms per 20 µL by pipetting 20 µL onto a glass slide or spare plate lid with a 200 µL pipette tip.
Add a magnetic stirrer bar to the glass bottle with the worms and place the bottle on the magnetic stirrer.
Change the volume of the ViaFill programme to 20 µL.
Prime the small tubing with the worms. As soon as liquid is clearly coming out of all 8 tubes, start dispensing worms into 96-well plates.
Check all wells to ensure worms were dispensed in all wells.
Incubate the plates at 20°C for 72h (Day 3)
Day 3.
After 72h since adding the worms to the plates, record the plates 4 wells at a time according to the specifications below using the Basler camera attached to a light microscope and the Basler software 'pylon Viewer' (6.2.4.9387).
Folder structure:
Save images in the correct folder:
- Ensure you have 400 GB free space on your computer.
- Copy the filepath to the folder on your local computer (e.g. /Users/jb3623/Desktop/basler/video/Exp2_N2/250226_72h/plate_2/A1_B2)
- For each new recording, change the subdirectory name from e.g. 'A1_B2' to 'A3_B4'.
Plate positioning:
It is essential that the plate is oriented according to the example below (starting with A1_B2).
Click on the 'scope' symbol indicated by the red arrow to activate the grid.
- Horizontal line crosses the sections where the top and bottom wells meet on both the left and the right side.
- Vetical line crosses the two wells on the right hand side just before the dark rings.
Recording Settings:
- Output format: Tiff
- Recording buffer size: 10 frame(s)
- Record a frame every: 5 Frame(s)
- Stop recording after: 10 seconds