TITLE: Reticulon Mutant Drug Screen
AUTHORS: Thomas J O'Brien

[DESCRIPTION]
Protocol for comparing the effects of various compounds on the phenotype our ret-1 (RTN2 ortholog) LoF mutant vs N2 control.

[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 case we are comparing the response of our ret-1 LoF mutant with N2 (parental control), therefore 20 x 90 mm plates will be picked in total.
SECTION: Prepare drug source plates (up to 3 days before first day of tracking)
2. Prepare 96-well source plates with stocks of all the compounds and concentrations to be tested. 

Note: It is best to shuffle around the position of compounds within the source plate to overcome any potential edge effects
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 (sterile water or DMSO) to achieve x1000 the highest concentration that will be tested (this accounts for dilution of compounds when adding to the tracking plates).

For example, to test at a compound at a maximum concentration of 100 µM, a 100 mM top stock of the compound is required
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 all desired concentrations to be tested.
SECTION: Prepare drug source plates (up to 3 days before first day of tracking)
2.4. Dispense 50 µL of the drug stocks into the relevant wells of a 96-well v-bottomed microtitre plate. Remember to include control wells (i.e. drug solvent only) and label the plates (unique plate ID and date) with a frost-proof marker pen.
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.

For this experiment 3 unique source plates were generated
SECTION: Pour 96-well tracking plates (up to 4 days before tracking)
3. Prepare 2 L no peptone NGM-agar and autoclave.

 

4. Once agar has cooled to around 62°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 (plate lids on) in an airtight container at 4°C.
 
For this experiment 24 tracking plates are imaged per day: pour 75 plates total
SECTION: Dry plates and bleach worms (5 days prior to tracking)
5. Dry 14 x 150 mm NGM-agar plates per strain (7 plates for ret-1 mutant and 7 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. Pre-label the 14 pre-seeded plates (prepared in step 5-6) with the relevant strain name and date, i.e. 7 x plates labelled "N2" and 7 x plates labelled "ret-1 LoF".
SECTION: Refeed L1s (3 days prior to tracking)
8.1. 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)
8.2. 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)
8.3. 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 of the pre-seeded/pre-labelled 150mm nursery plates.
SECTION: Refeed L1s (3 days prior to tracking)
8.4. 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)
8.5. Flip plates to be agar-side up and allow to grow at 20°C.
SECTION: Dry, dose and seed tracking plates (1 day prior to tracking)
9. In the morning, remove 24 square well 96-well plate from the cold room (prepared in steps 3-4) and weigh three random plates without their lids

Note: Number of plates will depend on the experiment being conducted
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 ret-1_02_rep8, where:
- 'ret-1' is the strain to be tested
- '_02' is the unique drug source plate number
- '_rep8' 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 section 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 (~15 seconds) 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. ret-1_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 9 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 'Tom_Stock_Dilution' custom program, premix drug in the drug source plates [position A of VIAFLO], and then transfer 1 µL drug in DMSO to the diluted drug plates prefilled with water (step 15) [position B of VIAFLO].

This creates a 1:10 dilution of the master compound stock, i.e. 100 mM -> 10 mM drug in diluted source plate
SECTION: Dry, dose and seed tracking plates (1 day prior to tracking)
17. Repeat for all drug stock plates.
SECTION: Dry, dose and seed tracking plates (1 day prior to tracking)
18. 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)
19. Use VIAFLO on the 'Tom_Agar' custom program to transfer 2.05 µL of diluted drug and water mixture onto the corresponding pre-labelled tracking plate [position A of VIAFLO].

This uses the formula below to perform a further 1:100 dilution of the compound, i.e. 10 mM diluted drug stock -> 100 µM final imaging plate concentration:

  

SECTION: Dry, dose and seed tracking plates (1 day prior to tracking)
20. Repeat steps 18-19 until all tracking plates have been dosed with the relevant compounds.



21. Ensure optical density of OP50 bacterial stock is ~1.0 (an OD of 0.96 - 1.04 is acceptable).
22. Use the VIAFILL dispenser to seed all the tracking plates with 5 µL bacterial suspension per well and leave to dry (~20 mins).
23. Flip tracking plates to be agar-side up, cover with an opaque box and leave at room temp overnight.
SECTION: Tracking
24. 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
25. Centrifuge using program 1 (1500 rpm for 2 mins) to pellet the worms.



SECTION: Tracking
26. Carefully remove and discard the supernatent and refill with M9 .
SECTION: Tracking
27. Repeat steps 25-26.
SECTION: Tracking
28. 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
29. Use COPAS wormsorter to dispense 3 worms per well (pure, no double) into 5 imaging plates at a time.
 

SECTION: Tracking
30. 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
32. Track the behaviour of worms incubated on the drugs 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

4 hour exposure to drugs:
- Place in tracking room at 13:50
- Start imaging run at 14:20
SECTION: Tracking
31. Use a microscope to check that wells are dry and incubate, agar-side up, in the 20°C incubator.
SECTION: Tracking
33. 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: Tracking
34. Transfer videos to BehavGenom and analyse with Tierpsy.