A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans

NOTE: Carry out all steps under sterile conditions (laminar flow cabinet) and with pre-sterilized materials (by autoclaving 126 °C, 11 min). An LB plate with streaked out E. coli OP50 kept at 4 °C is required, streak out onto fresh LB plates and restreak every month.

1. Bacterial Food (Escherichia coli OP50) Preparation

1. Day 1. Inoculate 2 x 5 ml LB in two universal bottles with a single colony of E. coli OP50 and place in shaking incubator at 37 °C (220 rpm) for 8 hr.
2. After 8 hr incubation, use 2 ml of E. coli OP50 LB culture to inoculate each of 3 x 200 ml LB. Place flasks in shaking incubator (220 rpm) O/N (17 hr) at 37 °C.
3. Weigh 20 x 50 ml centrifuge tubes and write weight on the tube.
4. Day 2. Using a serological pipette, aliquot 30 ml of O/N E. coli OP50 culture into the pre weighed centrifuge tubes. Centrifuge at 7,741 x g, 10 °C, 8 min.
5. Carefully decant the supernatant, keep the tube inverted with lid on and leave to stand for several minutes. Using a pipette remove any excess supernatant that may have collected in the lid. Weigh the tube and calculate the weight of the pellet.
6. Calculate the volume needed to provide a suspension of 30 g/L and mark this volume on the tube. Date, label and place tubes at -20 °C for use within 1-3 months or at – 80 °C if storing for longer than 3 months.
7. Prepare bacterial suspension for growing nematodes; allow bacterial pellet to thaw out and add required volume of S complete medium^11,12 to each tube, vortex gently to resuspend pellet, pool contents of different tubes to obtain the volume required. Work under sterile conditions.

2. Preparation of Drug Standards in a 96-well Plate Format

NOTE: If using a drug library, drug plates are provided at a single concentration of compound in DMSO. Primary screening will test compounds at a single concentration. Instructions follow for preparation of drug plate for confirmatory compound testing at a range of concentrations between 0-160 μM, selected after statistical significance at 10 μM. The steps below can be adapted to test other concentrations.

CAUTION! Follow necessary precautions for handling drugs (generally face mask, safety goggles, gloves are required).

1. Prepare drug plate with working standards for confirmatory screening: weigh the required amount of compound into sterile 1.7 ml microcentrifuge tube and prepare 16 mM compound in DMSO (i.e., 100x concentrated relative to desired top concentration for exposure).
2. Serially dilute 16 mM stock 1:2 in DMSO to obtain 8, 4, 2, 1, 0.5 and 0.25 mM standards (sterile conditions). These are 100x concentrated and will be diluted down to final concentrations of 0 (vehicle only), 2.5, 5, 10, 20, 40, 80 and 160 μM (in 1% DMSO) during drug exposure.
3. In a laminar flow cabinet, place 20-50 μl per well of compound standards within a column of a 96-well plate, for example plate position A1: 16 mM, B1: 8 mM, C1: 4 mM, D1: 2 mM, E1: 1 mM, F1: 0.5 mM, G1: 0.25 mM drug and H1: DMSO. Use different columns for dilution series of different drugs.
4. Aliquot vehicle to the wells of column 12 in the drug plate.
   NOTE: This will facilitate testing of vehicle control down columns in addition to the testing along row H, ensuring that vehicle is tested in positions representative of the whole plate.
   NOTE: Each drug plate for confirmatory screening holds dilution series for a maximum of 11 different drugs to be tested plus the vehicle control.
5. Label and seal plate, cover with foil and place at -20 °C until required.

3. Nematode Experiments

NOTE: Carry out all steps under sterile conditions, aseptically and with pre sterilized materials and reagents. Maintain C. elegans strains routinely on NGM plates with E. coli OP50.¹² Suggested timings for the different protocol steps are provided in Table 1.

1. Day 1 task: set up liquid culture of biosensor strain for subsequent synchronization.
   1. Take up nematodes from one 6 cm NGM plate (with plenty of young larvae where food has just run out) in 2 ml S complete and transfer to flask with 30 g/L E. coli OP50 in S complete (total volume 30 ml in a 250 ml capacity conical glass flask). Incubate 3 days at 20 °C, 160 rpm.
2. Day 4 task (allow approximately 30-35 min): bleach culture to harvest eggs and synchronize worm population. Carry out all centrifugation steps for 1 min, 600 x g.
   1. Prepare bleach solution just before use: to each 16 ml of 0.156 M KOH/NaOH add 4 ml bleach.
   2. Pour 2 x 14 ml worm culture into 15 ml conical centrifugation tubes. Allow worms to settle by gravity (3 min). Remove and discard liquid above settled nematodes. Add 5 ml of bleach solution to each tube and start timer. Combine volumes into one tube.
   3. Invert tube gently for 2 min, check under stereoscope for the breaking of worms and the release of eggs into suspension. When most eggs are released or at a maximum time of 2 min in the bleach solution, centrifuge.
   4. Remove supernatant carefully. Wash pellet 1x with 14 ml S complete and centrifuge.
   5. Discard supernatant, add 10 ml bleach solution and start timer (this second bleach step ensures that most of the worm carcasses disintegrate and are no longer seen under stereoscope). After a maximum time in bleach solution of 2 min, centrifuge.
   6. Wash pellet 3x in S complete, gently decant supernatant and resuspend pellet in 14 ml S complete after each centrifugation. After final wash, resuspend egg pellet in 14 ml S complete.
   7. Transfer the volume to a conical glass flask. Incubate 18-24 hr at 20 °C, 160 rpm.
      NOTE: Harvested eggs will hatch O/N and arrest development at the first larval instar (L1) due to lack of food, hence they will all be at the same stage of growth.
3. Day 5 task: set up synchronized worm cultures for drug assays.
   NOTE: Worms suspended in starvation medium tend to adhere to hydrophobic plastic surfaces such as pipette tips and tubes. 0.01% tween-20 is a surfactant used here to render plastic surfaces more hydrophilic and allow greater accuracy in counting (0.01% triton-X100 can also be used). When nematodes are in the bacterial supplemented medium, they don’t tend to stick to plastic.
   1. Determine numbers of hatched L1’s in flask, keep flask on shaking platform (160 rpm).
      1. Take 3x 100 μl samples from flask (use a clean tip each time) into separate 1.7 ml microtubes with 900 μl liquid medium 0.01% Tween-20.
         NOTE: Aspire 100 μl sample into the clean tip only once. Then when dispensing, pipette up and down a few times into the tween supplemented medium to release any worms adhering to the tip.
      2. Count the nematodes in 4x 10 μl droplets from each triplicate dilution tube. Calculate average value and estimate the number of worms present in the conical glass flask.
   2. Calculate the volume of hatched nematode suspension required to set up a 2 x 20 ml culture with 10 nematodes per 10 μl. Increase calculated volume by 10% to account for some subsequent nematode loss during centrifugation steps.
   3. Decant volume required into 2 x 15 ml centrifugation tubes (pre-weighed). Weigh tubes to obtain actual volume dispensed, vortex gently and adjust to target volume by removing excess volume with a 5 ml pipette (only the sterile tip should enter the tube).
   4. Make up volume to 14 ml with fresh S complete to wash nematodes. Centrifuge (1 min, 600 g). Remove and discard supernatant with care not to disturb the nematode pellet.
   5. Add 5 ml of S complete with 30 g/L E. coli OP50 to pelleted nematodes. Transfer the 5 ml in each tube to a conical glass flask containing 15 ml S complete with 30 g/L E. coli OP50. Note down time nematodes were first provided with food.
   6. Shake flask gently (160 rpm), take 9 x 10 μl droplets onto microscopic slides (use fresh tips each time) to count nematodes and confirm average of approximately 10 (±2) per 10 μl.
   7. Place nematode flasks in shaking incubator at 20 °C, 160 rpm for 42-44 hr.
4. Day 7 task: transfer nematodes to 96 well plates and initiate drug exposure.
   1. Combine nematode cultures into a single flask, keep swirling flask gently and place 3 x 4 ml of nematode culture in a 60 ml sterile trough. Place trough on a shaking platform (160 rpm).
   2. Use 8 channel pipette to aliquot 25 μl suspended nematodes per well of 96 well black microtiter plates with a flat transparent bottom (for taking both luminescence and fluorescence readings; or white plates for luminescence only). Cover with plate lid and set aside.
      1. After set up of every 2 plates, place another 2 x 2.5 ml nematodes in trough to replace lost volume (keep a ‘dead volume’ in trough of approximately 7 ml).
      2. Set up 13/14 plates with nematodes. 11 nematode plates will be required to test two 96-well drug plates. The 12^th nematode plate will be loaded exclusively with vehicle as a control. The 13^th plate will be used to establish background fluorescence on day 8 (see below). An extra plate can be prepared for use should mistakes occur during set up.
   3. Aliquot 74 μl of S complete to each well and place plate in a damp chamber (see materials list) in a shaking incubator (20 °C, 160 rpm) until ready to aliquot test compounds at the preselected developmental time (e.g., 45 hr 30 min after food provided). [Volume per well is now 99 μl].
   4. Thaw out drug plate(s) to test.
   5. Use multichannel pipette to set up nematode plates with drug, changing tips each time. Allow 5 min per 96 well plate for aliquoting drug.
      1. Take 1 μl from 1^st column of drug plate and add to columns 1-5 of a plate containing nematodes; repeat from 2^nd column in the drug plate into columns 8-12 of the plate containing nematodes. Add 1 μl of vehicle to columns 6 and 7 of the nematode plate.
         NOTE: The total volume per well in the nematode plate will be 100 μl resulting in a 1:100 dilution of compound/vehicle.
      2. Repeat process by adding 1 μl of the 3^rd/ 4^th column of the drug plate to columns 1-5 / 8-12 of the second nematode plate; add vehicle to columns 6 and 7 of the nematode plate.
      3. Continue to test remaining drug plate columns. Set up a minimum of one nematode plate with vehicle in all wells by sampling from column 12 of drug plate. Place plates back in damp chambers in shaking incubator (20 °C, 160 rpm) for 20-22 hr (see note for day 8).
   6. Part-prepare the luminescence buffer for next day: add DMSO and 10% Triton-X-100 to the required volume of S complete to obtain 1% DMSO and 0.15% Triton X-100. Allow 5 ml per plate read for luminescence, plus 1 ml for priming luminometer injector.
5. Day 8 task: Read experimental endpoints — fluorescence and bioluminescence. (GFP fluorescence readings are recommended as a means to normalize bioluminescence data.)
   NOTE: Aim to read endpoints by 66-67 hr after food provided to nematodes in order to prevent significant progeny production under the experimental conditions described.
   1. Set up plate to use as background control for GFP readings. Combine well contents from 13^th plate into a 15 ml tube. Allow nematodes to settle (2-3 min). Use supernatant to load a microplate (black with transparent bottom) with 100 μl bacterial suspension per well.
      1. Observe background control plate under microscope noting down wells where nematodes can be seen. Exclude these from the background estimate used in subsequent data analysis.
   2. Read fluorescence of each 96-well plate, including the background control. (Settings: optics from bottom of plate; filters 485/20 excitation; 528/20 emission.)
   3. Prepare buffer for luminescence readings: add luciferin (20 mM) to part-prepared buffer (from the previous day), to obtain luminescence buffer with 1% DMSO (1x), 0.15% Triton-X100 (3x) and 0.3 mM luciferin (3x). Prime luminometer injector 6x with 150 μl luminescence buffer.
      1. Previous step assumes 1% DMSO as vehicle during drug exposure. When vehicle is water, adjust the concentration of DMSO in luminescence buffer to 3% (i.e., 3x).
   4. Work with one plate at a time. Dispense 50 μl luminescence buffer per well. (150 μl final volume in well; 3x dilution of luminescence buffer: 1% DMSO, 0.05% Triton-X100 and 0.1 mM luciferin final concentrations.) Place immediately on shaking platform and start timer.
      1. After 3 min on shaking platform (160 rpm), read luminescence (1 sec per measurement).

4. Data Analysis

1. Subtract average GFP background reading from fluorescence results. Divide bioluminescence by respective GFP reading.
   1. When a high GFP value is detected for nematodes exposed to a compound, measure fluorescence of the compound on its own to account for any compound fluorescence. If higher than average, use this as the background instead.
2. Express bioluminescence, GFP normalized bioluminescence and GFP fluorescence data as a percentage of the average value for vehicle control in each plate.
3. Plot average values and error bars for each concentration.
4. Test for statistical significance using 2-way analysis of variance (ANOVA) with effects of ‘Concentration’, ‘Experiment’ and their interaction (each data point refers to a single well), and use the Dunnett test as post-hoc test (vs. vehicle control).
   NOTE: If the “experiment” or the “interaction” terms are significant, further experiments may be required to confirm response. Where no variability between experiments is apparent from observation of data plots, a One-way ANOVA can be performed on pooled data from independent experiments.
   1. For the statistical analysis of plate(s) with vehicle only, select all wells in columns 6 -7 and all wells in row H as control group (these are the positions routinely assigned to vehicle during compound testing).