Feb 22, 2022
Keio Acute Response Antioxidant Rescue
- 1Imperial College London
Protocol Citation: Saul Moore 2022. Keio Acute Response Antioxidant Rescue . protocols.io https://dx.doi.org/10.17504/protocols.io.b3nnqmde
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: January 10, 2022
Last Modified: February 22, 2022
Protocol Integer ID: 56750
Abstract
Phenotyping the acute behavioural response of Caenorhabditis elegans (N2 Bristol) to E. coli single-gene deletion mutants (BW25113), in both the presence and absence of antioxidants (Trolox, NAC, vitamin C and resveratrol).
Videos are recorded at 25 fps on the laboratory’s (Hydra) imaging rig, immediately after worms are picked onto imaging plates, for a total of 45 minutes at 25 fps, with blue-light stimulus delivered for 10 seconds in 5 minute intervals throughout the recording.
Guidelines
Read through the whole protocol before starting, as the sections are not in chronological order
Materials
10 x 6-well plates (imaging plates)
10 x 60mm Petri plates (maintenance plates
3 x 90mm Petri plates (nursery plates)
For 1L NGM agar:
- 3g NaCl (ref: fisher 447300010 )
- 2.5g bactopeptone (ref: BD 21167 lot 8270639)
- 17g agar (ref : A7002-5KG lot BCBM1702V)
- 1L H2O
- Salts (post-autoclave):
25mL KH2PO4, pH=6.0 (ref : SIGMA P0662-2,5KG)
1mL MgSO4, 7H2O (1M) (ref : SIGMA M5921-500G)
1mL CaCl2 (1M) (ref : SIGMA C5080-500G)
1mL cholesterol (5mg/mL) (ref : SIGMA C8667-5G)
For 1L LB broth:
- 25g LB powder (ref : fisher BP9723-500)
- 1L H2O
For 1L M9:
- 3g KH2PO4
- 7g Na2HPO4.2H2O (ref : SIGMA 71645-1KG )
- 5g NaCl
50mg/mL Kanamycin (in water, filter 0.2mm)
Antioxidants:
Trolox, NAC, Vitamin C:
- Initial concentration: 1mg/mL
- Final concentration : 200mg/mL in 4mL M9
- (pH for Trolox adjusted with NaOH)
Trans-resveratrol:
- Initial concentration: 100mg/mL 200mg in 2ml ethanol (in the freezer -20)
Intermediate concentration : 1mg/ml à in 100mL in 10ml M9 (filtered sterile)
Final concentration : 200mg/mlà in 4ml of M9 sterile, put 800ul of the intermediate solution (1mg/ml)
Put 200ul on plate (4ml NGM) to have a concentration of 10ug/ml
Trolox 238813-1G sigma (in DMSO first)
Trans resveratrol : PHR2201-200MG sigma (dilution in DMSO)
NAC, Vitamin C (dilution in water)
Safety warnings
Goggles must be worn when operating high-power blue-light LEDs on the rig
Before start
Prepare NGM agar media and fill maintenance plates for raising N2 Bristol C. elegans on BW25113 background strain
Preparing maintenance plates
Preparing maintenance plates
1d
1d
Make 500mL normal Nematode Growth Media (NGM) agar, following the protocol
Protocol
NAME
Making normal NGM for imaging plates (Cabreiro Lab)
CREATED BY
Saul Moore
2h
Under a hood, pour 20ml NGM agar into each of 10 x 60mm Petri plates (maintenance plates), and leave to dry for apprximately 1 hour. Once dry, store at 4°C until seeding bacterial lawns
1h 30m
Inoculate an overnight culture of E. coli BW25113 bacteria in an Erlenmeyer flask containing 50ml LB broth, and place in at 37°C in a shaking incubator (200rpm)
Protocol
NAME
Inoculating a Liquid Bacterial Culture
CREATED BY
Priota Islam
20h
Remove the BW culture from the shaking incubator and store at 4°C until seeding
Remove the maintenance plates and the BW25113 culture from 4°C storage, and leave on the bench for approximately 30 minutes to acclimate to room temperature
30m
Seed the maintenance plates each with 200μL of BW25113 culture, using aseptic technique working under a microbiological hood
Leave under hood until dry (approximately 30 min to 1 hour timing depends on humidity)
Preparing worms
Preparing worms
2d
2d
Using a platinum pick, gently pick 30 L4-stage N2 Bristol C. elegans onto each maintenance plate, and store in an incubator at 20°C (Monday)
1d
After 24 hours, remove the adult worms, leaving the eggs behind to hatch into L1 larvae (Tuesday)
1d
Bleach-synchronise the worms by performing an egg prep, following the protocol:
(Friday)
Protocol
NAME
Egg Prep for Bleach Synchronization (Cabreiro Lab)
CREATED BY
Saul Moore
At around noon the next day, wash L1 larvae off the empty plate with a few mL of M9 using a glass Pasteur pipette, and re-feed onto BW-seeded maintenance plates. Incubate at 20°C.
(Saturday)
Preparing bacteria
Preparing bacteria
Prepare two Erlenmeyer flasks with 50mL LB for inoculating a liquid bacterial culture of BW control and BW25113ΔfepD bacteria. Add 50mg/mL Kanamycin to BW25113ΔfepD flask
(Friday)
Inoculate from a single colony picked from streaked LB plates stored at 4°C, following the protocol:
Protocol
NAME
Inoculating a Liquid Bacterial Culture
CREATED BY
Priota Islam
Leave overnight in a shaking incubator (37°C, 200rpm)
The next day, remove the bacterial cultures from the shaking incubator, and prepare another two Erlenmeyer flasks each with 50mL LB, for inoculating a second round of overnight cultures. Only this time, do not add Kanamycin.
(Saturday)
Inoculate the second round of cultures by pipetting approximately 50μL bacterial culture from the first overnight culture. Leave in a shaking incubator overnight (37°C, 200rpm)
The next day, remove the overnight cultures from the shaking incubator, and store at 4°C until seeding imaging plates the following day
(Sunday)
Preparing imaging plates
Preparing imaging plates
Make 250mL normal Nematode Growth Media (NGM) agar, following the protocol in go to step #1
(Thursday)
Safety information
Wait for the agar to cool to approximately 55°C before adding the salts, to avoid 'speckling' in imaging plates
Pour 3.5mL NGM agar into each well of 10 x 6-well plates, and leave to cool under a hood. Then store at 4°C until seeding lawns
Remove imaging plates from 4°C, and dry under a hood for 30 minutes to remove condensation. Remove the bacterial cultures from 4°C and leave on the bench for 30 minutes to acclimate to room temperature.
(Monday)
Pipette 30μL of bacterial culture into the centre of each well in the 6-well imaging plates, taking care not to damage the agar with the pipette tip. Seed half of the 6-well plates with BW control, and the other half with BW25113ΔfepD lawns
Leave the seeded plate to dry for 20 minutes under the hood, then transfer to a 25°C incubator and leave to grow for a further 7 hours and 40 minutes (for a total of 8 hours lawn growth time), before storing at 4°C for tracking the next day
On the day of tracking, remove the seeded plates from 4°C and dry under a hood for 30 minutes to remove condensation
(Tuesday)
Prepare the antioxidants (see Materials section for details of antioxidant preparation). Pipette 200μL antioxidant solution onto the top of the lawns of each well, to yield a final concentration of 10μg/mL antioxidant (in 4mL NGM agar). Leave under a hood to dry for approximately 30 minutes
Leave the plates for at least 2 hours after adding antioxidants before picking worms onto the plate and tracking
Hydra Tracking
Hydra Tracking
Prior to tracking, ensure that the imaging cave air conditioning is turned on (and there has not been a power-cut) and also empty the dehumidifier waste water tray (see pre-imaging checklist)
(Tuesday)
Safety information
Normal temperature range: 19 - 21°C
Humidity: 35 - 45%
Remove the seeded imaging plates from 4°C and leave on bench top (with lids on) for 30 minutes to acclimate to room temperature
Remove the plate of age-matched (Day1 adult) worms from 20°C incubator
Using an eyebrow hairpick, gently but swiftly transfer 10 worms onto the edge of the bacterial lawn of each of 6 imaging plates
Quickly transport the 6-well plates to the imaging cave and place them under the rigs (recording the respective camera positions of each of the wells in the metadata along with the relevant treatment information)
Track worm behaviour on the different food + antioxidant treatment conditions for a total of 45 minutes (25 fps), applying a 10-second bluelight stimulus every 5 minutes