TITLE: Response of Disease Model Mutants to Cholinergic Drugs
AUTHORS: Thomas J. O'Brien

[DESCRIPTION]
Protocol for testing if disease model mutant C. elegans strains show increased resistance/sensitivity to the action of cholinergic drugs after 1 and 4 hour exposure times.

[GUIDELINES]
For the 1 hour time point it is important to get plates into the incubator with the lids off as quickly as possible to allow the wells to dry out prior to imaging.

[STEPS]
SECTION: Pick L4 worms for bleaching (9 days prior to tracking)
1. Pick 10 x L4 worms onto 4 x 90mm NGM-agar plates pre-seeded with E. coli OP50 for each strain (remember to include a relevant control strain).

e.g. if looking to compare the response of one mutant to N2 reference: you would have 4 x 90 mm plates with strain 1 and 4 x 90 mm plates with N2
SECTION: Prepare drug source plates (up to 3 days before first day of tracking)
2. Prepare 1 x 96-well source plate per compound to be tested
SECTION: Prepare drug source plates (up to 3 days before first day of tracking)
2.1. Weigh out compounds using a micro balance and dissolve in an appropriate  solvent to achieve x1000 the highest concentration that will be tested (this accounts for dilution of compounds when adding to the tracking plates)

e.g. here compounds were tested at a maximum concentration of 100 µM, therefore make a 0.1 M top-stock
SECTION: Prepare drug source plates (up to 3 days before first day of tracking)
2.2. Perform a 10-fold serial dilution of the top-stock of each compound, using the same solvent initially used to dissolve the compounds 

For example: mix 50 µL 0.1 M compound top stock with 450 µL solvent and vortex (yielding 0.01 M concentration)
SECTION: Prepare drug source plates (up to 3 days before first day of tracking)
2.3. Repeat previous step (using the newly generated dilute compound) to obtain 3 x concentrations of drugs (in this case 0.1, 0.01 and 0.001 M)
SECTION: Prepare drug source plates (up to 3 days before first day of tracking)
2.4. Dispense 30 µL of the drug stocks into the relevant wells of a 96-well round bottomed micro-titre  plate. Remember to include control wells (i.e. the drug solvent only) and label the plates with a frost-proof marker pen.

For this experiment I dispensed each drug concentration into 2 rows of a plate. Allowing 12 well replicates for the control and 12 well replicates for the mutant strain per compound, per concentration. This layout also allowed me 2 x rows for the solvent only controls.
SECTION: Prepare drug source plates (up to 3 days before first day of tracking)
2.5. Use a plate seal to seal source plates, and store at -20°C until use 
SECTION: Pour 96-well tracking plates (up to 4 days before tracking)
3. Prepare 1 L no peptone NGM-agar and autoclave

 

SECTION: Pour 96-well tracking plates (up to 4 days before tracking)
4. Once agar has cooled to around 65°C , add the post-autoclave salts and cholesterol then dispense agar into square 96-well plates using VIAFILL dispenser. Dispense 200 µL  per well. Once cooled, store agar side up in an airtight container at 4°C

(For this experiment 4 drugs were tested across three independent imaging days on two disease model mutant strains, totalling 24 tracking plates) 
 

SECTION: Dry plates and bleach worms (5 days prior to tracking)
5. Dry 2 x 150 mm NGM-agar plates per strain (including 2 plates for N2) in the drying cabinet (setting 2) for 3 hours
SECTION: Dry plates and bleach worms (5 days prior to tracking)
6. Seed 150 mm plates with E. coli OP50 and leave to dry on bench at room temperature overnight
SECTION: Dry plates and bleach worms (5 days prior to tracking)
7. Bleach synchronise worms prepared in step 1 and leave in diapause for 2 days at 20°C on a rotator that is constantly spinning

 

SECTION: Refeed L1s (3 days prior to tracking)
8. At 16:45, spin L1s (prepared in step 7) for day 1 of tracking using centrifuge program 1 (1500 rpm for 2 mins)
SECTION: Refeed L1s (3 days prior to tracking)
8.1.  Use a 3 mL plastic pasteur pipette to carefully remove the supernatant (leaving ~0.5 mL) then resuspend the worm pellet by gently flicking the tube

SECTION: Refeed L1s (3 days prior to tracking)
8.2. Using a clean rubber teat and glass pipette per strain, carefully aspirate the L1 worm suspension and drop 4 small droplets around the edges of the pre-seeded 150 mm plates (prepared in step 6)

[This will yield 2 x 150 mm plates of refed L1s per strain being studied + controls]
SECTION: Refeed L1s (3 days prior to tracking)
8.3. Allow to grow at 20°C
SECTION: Dry, dose and seed tracking plates (1 day prior to tracking)
9. In the morning, remove 1 square well 96-well plate from the cold room per strain and compound to be tested and weigh three random plates without their lids

(Note: If using the layout suggested above each plate will contain reference and control strains. Hence, if testing 2 x strains and 4 x compounds, remove 8 plates from the cold room)
SECTION: Dry, dose and seed tracking plates (1 day prior to tracking)
10. Allow plates to dry, with lids off, in the drying cabinet (setting 2) until they have reduced in weight by 3-5% (~1.5 - 2 hours)
SECTION: Dry, dose and seed tracking plates (1 day prior to tracking)
11. Pre-label dried tracking plates (step 10) with the imaging run and drug information, so that every plate on a single day of imaging has a unique plate id. For example aldicarb_hlb-1_01, where:
- 'aldicarb' is the drug to be tested
- 'hlb-1' is the strain of interest
- '01' is the imaging run number that day
SECTION: Dry, dose and seed tracking plates (1 day prior to tracking)
12. Remove drug source plates (made in step 2) from the -20 °C freezer and allow to thaw at room temperature
SECTION: Dry, dose and seed tracking plates (1 day prior to tracking)
13. Briefly spin drug source plates in the mini bench-top plate spinner to collect all plate contents at the bottom of the wells
SECTION: Dry, dose and seed tracking plates (1 day prior to tracking)
14. Pre-label skirted 96-well PCR plates with drug source plate information (e.g. aldicarb_01) that will be used to make up the diluted drug plates
SECTION: Dry, dose and seed tracking plates (1 day prior to tracking)
15. Next to a flame, dispense 7 µL of sterile water into each well of the pre-labeled dilution plates (step 14) using multichannel pipette and reagent reservoir

SECTION: Dry, dose and seed tracking plates (1 day prior to tracking)
16. Using VIAFLO multi-well dispenser on the 'BG_STOCK' custom program, premix drug in the drug source plates [position A of VIAFLO], and then transfer 0.5 µL drug in DMSO to the diluted drug plates prefilled with water (step 15) [position B of VIAFLO] -- Repeat for all drugs

SECTION: Dry, dose and seed tracking plates (1 day prior to tracking)
17. Working one plate at a time (so that plates don't dry before drugs are added), use the VIAFILL (with the small  cassette) to dispense 5 µL of sterile deionised  water onto a pre-dried imaging plate (step 10)
SECTION: Dry, dose and seed tracking plates (1 day prior to tracking)
18. Use VIAFLO on the 'BG_AGAR' custom program to transfer 3 µL of diluted drug and water mixture onto the corresponding pre-labelled tracking plate [position A of VIAFLO]

SECTION: Dry, dose and seed tracking plates (1 day prior to tracking)
19. Repeat steps 17-18 until all tracking plates have been dosed with the relevant compounds
SECTION: Dry, dose and seed tracking plates (1 day prior to tracking)
20. Prepare 1:10 dilution of OP50 in M9 in a small, sterile bottle:
- 5 mL OP50
- 45 mL M9
SECTION: Dry, dose and seed tracking plates (1 day prior to tracking)
21. Use the VIAFILL dispenser to seed all the tracking plates with 5 µL bacterial suspension per well

SECTION: Tracking
22. At 09:00, wash young adult worms off the 150 mm plates using M9 buffer, and a clean 3 mL pasteur pipette per strain, into  15 mL falcons.
SECTION: Tracking
23. Centrifuge using program 1 (1500 rpm for 2 mins) to pellet the worms
SECTION: Tracking
24. Carefully remove and discard the supernatent and refill with M9


SECTION: Tracking
25. Repeat steps 23-24
SECTION: Tracking
26. After final wash, fill 15 mL falcon with M9. Transfer contents to a clean 50ml falcon and top up to 45 mL with M9
SECTION: Tracking
27. Use COPAS wormsorter to dispense 3 worms per well (pure, no double) into 5 imaging plates at a time

 

SECTION: Tracking
28. Allow liquid to dry off by placing imaging plates in 20°C incubator with lid off for 30 minutes
SECTION: Tracking
29. Expose worms to the drugs for 1 and 4 hours, as calculated from the middle wormsorter time and allow worms to acclimate for 30 minutes in the cave prior to imaging. Remember to return plates, agar-side up with lids on, to the 20°C incubator in between the 1 and 4 hour time points.

For example:
- wormsorter start time 10 :00
- wormsorter end time 10:40
- middle wormsorter time 10:20

1 hour exposure:
- cave time = 10:50
- imaging start time = 11: 20

4 hour exposure:
- cave time 11:50
- imaging start time 12:20
SECTION: Tracking
30. Image on the multi-camera tracker (Hydra) using 'run_syngenta_experiment_v2.py' protocol script (5 mins prestim; 6 mins bluelight with 60 sec OFF, [10sec ON, 90sec OFF] x 3 times; 5 mins postsim)
SECTION: Tracking
31. Transfer videos to BehavGenom and analyse with Tierpsy