TITLE: Screening effects of excess copper on worm behaviour
AUTHORS: Thomas J O'Brien

[DESCRIPTION]
Protocol for tracking the behaviour of mutant (cua-1[H828Q], Wilson's Disease ortholog) vs wild-type (N2) worms: in the absence of treatment; exposed to 25-100uM CuCl₂ for 4h; reared for one generation on NGM+CuCl₂ plates (F1s); and reared for two generations on NGM+CuCl₂ plates (F2s).

[STEPS]
SECTION: Pick L4 worms for bleaching (9 days prior to tracking)
1. Pick 10 x L4 worms onto 10 x 90mm NGM-agar plates pre-seeded with E. coli OP50 for each strain to be tested. In this protocol we are comparing the response of a mutant strain containing a H828Q mutation in the cua-1 gene, cua-1[H828Q], to WT control (N2). 

Therefore 20 x 90 mm plates will be picked in total. 
SECTION: Pour 150mm nursery plates (up to 6 days prior to tracking)
2. Prepare 4.5L NGM-agar and autoclave.
 

SECTION: Pour 150mm nursery plates (up to 6 days prior to tracking)
3. Meanwhile prepare a 1 M stock solution of copper chloride (CuCl₂) by dissolving 134.45mg of powder in 1mL of sterile deionised water.

   

SECTION: Pour 150mm nursery plates (up to 6 days prior to tracking)
4. Sterilise by passing through a 0.22 micron syringe filter into a clean microfuge tube wrapped in tinfoil.
SECTION: Pour 150mm nursery plates (up to 6 days prior to tracking)
5. Once agar has cooled to around 62°C , add the post-autoclave salts and cholesterol (as described in the protocol above).
SECTION: Pour 150mm nursery plates (up to 6 days prior to tracking)
6. Label 4 x pre-sterilised 1L bottles (red screw cap lids from media kitchen) with desired concentration of CuCl₂ to be tested (i.e. 100;75,50;25µM).

Aseptically transfer 1L of NGM to each of these bottles. I recommend doing this in the microbial hood that has been throughly wiped down with 70% EtOH/Distel prior to use.
SECTION: Pour 150mm nursery plates (up to 6 days prior to tracking)
7. Aseptically add CuCl₂ from stock to desired concentration, if preparing 1L solutions and 1M stock (as above):
100uL stock solution [1M] in 1L NGM = 100µM final conc
75uL stock in 1L = 75µM
50uL stock in 1L = 50µM
25uL stock in 1L = 25µM
SECTION: Pour 150mm nursery plates (up to 6 days prior to tracking)
8. Here we want to look for behavioural differences between the mutant and control for the first generation (F1) and second generation (F2) worms reared on various concentrations of copper. Since we will be doing 3 day replicates, we will need 12 x 150 mm plates for each concentration to be tested. 

Hence, pre-label 12 x 150mm petri dishes (nursery plates) for each concentration of copper.

The remaining 0.5L of NGM will be used for 'no compound/no treatment' control plates (only 6 needed in total). 
SECTION: Pour 150mm nursery plates (up to 6 days prior to tracking)
9. Pour 75mL of agar into the respective nursery plates and leave to dry in the hood.
 

SECTION: Pour 150mm nursery plates (up to 6 days prior to tracking)
10. Once dry, invert plates to be agar-side up and place in an opaque/tin foil-lined air tight container. Store at 4°C. 
SECTION: Pour tracking plates (up to 4 days prior to tracking)
11. Prepare 1L of no-peptone NGM and autoclave.
 

SECTION: Pour tracking plates (up to 4 days prior to tracking)
12. Once agar has cooled to around 62°C , add the post-autoclave salts and cholesterol (as described above).
SECTION: Pour tracking plates (up to 4 days prior to tracking)
13. Meanwhile make up fresh stocks of 1M CuCl₂ (as described above).
SECTION: Dry plates and bleach worms (5 days prior to tracking F1 worms)
17. Dry 2 x 150 mm NGM-agar plates of copper concentration (i.e. 0;25;50;75;100µM, with 1 plates for mutant and control) in the drying cabinet (setting 2) for 3 hours.
SECTION: Pour tracking plates (up to 4 days prior to tracking)
14. Label 4 x pre-sterilised 250mL bottles (red screw cap lids from media kitchen) with the concentrations of CuCl₂ to be tested (i.e. 100;75,50;25µM).

As above, aseptically transfer 200mL of NGM to each of these bottles and add CuCl₂ to the desired final concentration.
SECTION: Pour tracking plates (up to 4 days prior to tracking)
15. Pre-label 9 x tracking plates with each concentration of CuCl₂. This will allow for 3 day replicates of: 4h exposure to excess copper, F1 worms and F2 worms to be tracked on separate plates.

Label 3 x tracking plates as 'NGM only' for the no compound controls. 
SECTION: Pour tracking plates (up to 4 days prior to tracking)
16. Dispense agar into square 96-well plates using VIAFILL dispenser. Dispense 200 µL  per well. Once cooled, store agar side up (plate lids on) in an opaque/tinfoil lined airtight container at 4°C.
 

SECTION: Dry plates and bleach worms (5 days prior to tracking F1 worms)
19. 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: Dry plates and bleach worms (5 days prior to tracking F1 worms)
18. Seed 150 mm plates with E. coli OP50 and leave to dry on bench at room temperature overnight.
SECTION: Refeed L1s (3 days prior to tracking F1 worms)
20. Pre-label the pre-seeded 150mm plates with the relevant strain name and date, i.e. 'N2' or 'cua-1[H828Q]', for each copper concentration, remember to include a no compound control.


SECTION: Refeed L1s (3 days prior to tracking F1 worms)
21. 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 F1 worms)
22. Use a 3 mL plastic pasteur pipette to carefully remove the supernatant (leaving ~1 mL) then resuspend the worm pellet by gently flicking the tube.


SECTION: Refeed L1s (3 days prior to tracking F1 worms)
23. At 17:00, use a clean rubber teat and glass pipette per strain, carefully aspirate the L1 worm suspension and drop 4 small droplets onto the bacterial lawn (~1000-1500 worms total) of the pre-seeded/pre-labelled 150mm nursery plates.


SECTION: Refeed L1s (3 days prior to tracking F1 worms)
24. Incubate, agar-side down, at 20°C for 15 mins to allow the droplets containing L1 worms to dry.
SECTION: Refeed L1s (3 days prior to tracking F1 worms)
25. Flip plates to be agar-side up and incubate at 20°C. 
SECTION: Dry and seed tracking plates (1 day prior to tracking F1 worms)
26. In the morning, remove 2 x square 96-well tracking plate from the cold room per copper concentration and weigh three random plates without their lids.

SECTION: Dry and seed tracking plates (1 day prior to tracking F1 worms)
27. 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 and seed tracking plates (1 day prior to tracking F1 worms)
28. 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 100µM_F1_run1, where:
- '100µM' is the concentration of copper on the plate
- '_F1' stands for F1 worms reared on copper from the point of refeeding (use '4h' for 4h exposure)
- '_run1' is the imaging run number that day
SECTION: Dry and seed tracking plates (1 day prior to tracking F1 worms)
29. Ensure optical density of E. coli OP50 stock is ~1.0 (an OD of 0.96 - 1.04 is acceptable).



SECTION: Tracking F1 worms and worms exposed to excess copper for 4h
33. 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 (labelled with strain name and copper treatment).

SECTION: Dry and seed tracking plates (1 day prior to tracking F1 worms)
30. Use the VIAFILL dispenser to seed all the tracking plates with 5 µL bacterial suspension per well and leave to dry (~20 mins).
SECTION: Dry and seed tracking plates (1 day prior to tracking F1 worms)
31. Flip tracking plates to be agar-side up, cover with an opaque box and leave at room temp overnight.
SECTION: Tracking F1 worms and worms exposed to excess copper for 4h
34. Centrifuge using program 1 (1500 rpm for 2 mins) to pellet the worms.
SECTION: Tracking F1 worms and worms exposed to excess copper for 4h
35. Carefully remove and discard the supernatent and refill with M9 .
SECTION: Tracking F1 worms and worms exposed to excess copper for 4h
36. Repeat steps 33-34.
SECTION: Tracking F1 worms and worms exposed to excess copper for 4h
37. 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 F1 worms and worms exposed to excess copper for 4h
38. Use COPAS wormsorter to dispense 3 worms per well (pure, no double) into their respective imaging plates, filling 5 plates at a time.
 

SECTION: Tracking F1 worms and worms exposed to excess copper for 4h
38.1. To test the effects of exposing worms to copper for 4h simply dispense worms from the NGM only nursery plates into the wells of tracking plates containing the desired concentration of copper. 

In this experiment I fill half of the plate with WT and half with cua-1[H828Q]
SECTION: Tracking F1 worms and worms exposed to excess copper for 4h
39. Allow liquid to dry by placing imaging plates in microbial safety cabinet (pre-sterilsed with 70% ethanol) with lids off for ~30 minutes.
SECTION: Tracking F1 worms and worms exposed to excess copper for 4h
40. Use a microscope to check that wells are dry and incubate, agar-side up, in the 20°C incubator.
SECTION: Tracking F1 worms and worms exposed to excess copper for 4h
41. Track worms incubated on the tracking plates after 4 hours, as calculated from the median point of dispensing worms (middle wormsorter time), and allow worms 30 mins to acclimatise to conditions in the tracking room prior to imaging. 

For example: 
- Wormsorter start time = 10:00
- Wormsorter end time = 10:40
- Middle wormsorter time = 10:20

- Place in tracking room at 13:50
- Start imaging run at 14:20
SECTION: Tracking F1 worms and worms exposed to excess copper for 4h
42. Image on the multi-camera tracker (Hydra) using 'run_syngenta_experiment_v2.py' protocol script: 

1) 5 min pre-stimulus recording
2) 6 min blue light recording: 60 sec no light, [10 sec light ON, 90 sec light OFF] x 3     (6 min total)
3) 5 min post-stimulus recording
SECTION: Preparing F2 worms (Day of tracking F1 worms)
43. While tracking plates are drying, recover the 50mL falcon tubes containing worms reared on various copper concentrations and transfer remaining worm suspension to fresh 15mL falcon tubes.
SECTION: Dry and seed F2 nursery plates (1 day prior to tracking F1 worms)
32. Prepare nursery plates for rearing the second generation of worms (F2) on increased copper concentrations by repeating steps 17-18. 

Again, use 1 x 150mm plate for WT and mutant strain. Ensure that you clearly labelled the plates with the concentration of copper, worm strain and generation number.
SECTION: Preparing F2 worms (Day of tracking F1 worms)
44. Centrifuge using program 1 (1500 rpm for 2 mins) to pellet the worms.
SECTION: Preparing F2 worms (Day of tracking F1 worms)
45. Use a 3 mL plastic pasteur pipette to carefully remove the supernatant (leaving ~1 mL) then resuspend the worm pellet by gently flicking the tube.
SECTION: Preparing F2 worms (Day of tracking F1 worms)
46. Refeed ~50 gravid worms onto fresh nursery plates containing the same concentration of copper they have been reared on. 

As before, allow droplets to dry and incubate, agar-side up, at 20°C overnight.
SECTION: Preparing F2 worms (1 day after tracking F1 worms)
47. The F1 worms refed yesterday will have now laid their eggs onto the F2 nursery plates. Use an eyelash pick to remove these adult worms from the plates, continue incubating the nursery plates at 20°C monitoring their development.
SECTION: Dry and seed tracking plates (1 day prior to tracking F2 worms)
48. Repeat steps 26-31 to prepare plates for tracking the behaviour of the second generation of worms reared on plates containing excess copper.
SECTION: Tracking F2 worms
49. Repeat steps 33-42 to track the F2 worms.
SECTION: Tracking F2 worms
50. Transfer videos to BehavGenom and analyse with Tierpsy.