TITLE: Disease model screen protocol
AUTHORS: Ida Barlow

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Protocol for screening the disease model </span><span style = "font-style:italic;">C. elegans</span><span> strains under baseline and bluelight conditions using the Hydra (Loopbio) imaging rigs. This is the protocol for one day of imaging and should be repeated at least three times to get replicates from three separate days. The number of imaging runs depends on the number of strains being screened and whether strains are sorted into 96 well plates row-wise (7 strains per plate) or column-wise (11 strains per plate).</span></div></div>

[STEPS]
?. [Pick L4 worms for bleaching (-10 days before tracking; eg Monday)]
Pick 5 x L4s per strain onto 3 x 60mm plates (pre-seeded with OP50) for each strain
?. [Pick L4 worms for bleaching (-10 days before tracking; eg Monday)]
Take out 90mm plates from cold room to allow to dry at room temperature
3 x 90mm plates per strain being imaged
?. [Pour 96WPs (anytime up to 2 days before tracking)]
Prepare appropriate volume of low peptone NGM and autoclave
How to calculate volume of agar requirednumber of imaging runs = (number of strains) / fill_rows (7) or fill_columns (11)number of plates = number of imaging runs * number of rigs (default=5 hydras)volume agar (mL) = number of plates * 96 * 0.2Round up the volume of agar required to the nearest half litreNB.fill_rows if filling plates with each row as a different strainfill_columns if filling plates with each column as a different strainevery plate must have a row/column saved for controls, so fill_rows/columns = number_columns/rows - 1
?. [Pour 96WPs (anytime up to 2 days before tracking)]
Once agar has cooled to around  , add the salts and dispense agar into 96 well plates using VIAFILL dispenser. Dispense   per well. Once cooled, store agar side up with one lid per stack of 5 plates in an airtight container at
65 °C
200 µl
4 °C
?. [Seed plates, bleach worms (-5 days before tracking eg Friday)]
Seed with OP50 the 90mm plates that have been drying at room temperature
?. [Seed plates, bleach worms (-5 days before tracking eg Friday)]
Bleach worms prepared for day 1 of tracking
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?. [Refeed L1 (-3 days before tracking eg Sunday)]
At 11:00 spin L1s at 2500rpm. Remove supernatent and using glass pipette, drop 3-4 small droplets around the edges of the plate (off food) onto 3 x seeded 90mm plates per strain.Allow to grow at
20 °C
?. [Dry and seed 96 well plates  (-1 days before tracking eg. Tuesday)]
Take required number of 96 well plates from the cold room, and weigh three random plates without their lids
?. [Dry and seed 96 well plates  (-1 days before tracking eg. Tuesday)]
Place in cabinet dryer (setting 2) and allow to dry for 2-3 hours with lids off
?. [Dry and seed 96 well plates  (-1 days before tracking eg. Tuesday)]
Weigh 3 random plates and verify that at least 3-5% reduction in weight
?. [Dry and seed 96 well plates  (-1 days before tracking eg. Tuesday)]
Prepare 1:10 dilution of OP50 in M9 in small bottle
20ml total volume is sufficient for 10 96 well plates
?. [Dry and seed 96 well plates  (-1 days before tracking eg. Tuesday)]
Using VIAFILL dispenser, seed all the imaging plates with  per well
5 µl
?. [Dry and seed 96 well plates  (-1 days before tracking eg. Tuesday)]
Leave plates overnight at room temperature with lid
?. [Imaging (eg Wednesday)]
Pre-label imaging plates (square well) with IR visible pen or labels
Use naming system:SXX_RYYXX = imaging run numberYY = rig (hydra number)
?. [Imaging (eg Wednesday)]
Wash one batch of worms (strains for imminent imaging run) off 90mm paltes with M9 buffer using pasteur pipette into 15ml falcons
?. [Imaging (eg Wednesday)]
Spin at 1500rpm for 2 minutes to pellet the worms
?. [Imaging (eg Wednesday)]
Remove supernatent and fill with M9
?. [Imaging (eg Wednesday)]
Repeat steps 18-19
?. [Imaging (eg Wednesday)]
After final wash, fill falcon with M9 and transfer contents from 15ml falcon to a 50ml and fill up 30ml with M9
?. [Imaging (eg Wednesday)]
Record start time for sorting this batch of worms
?. [Imaging (eg Wednesday)]
Use COPAS wormsorter to dispense 3 worms per well (pure, no double) into specified wells for 5 x 96WPs. Use pre-made YYYYMMDD_wormsorter.csv to plan and determine which strains to dispense into each row of each plate
?. [Imaging (eg Wednesday)]
Between each strain, wash with purified H2O by placing water falcon into sample cup and washing through by selecting Maintenance -> FlushSample
?. [Imaging (eg Wednesday)]
Once the plates for this imaging run have been filled, record the end time
?. [Imaging (eg Wednesday)]
Allow liquid to dry off by placing imaging plates in  incubator with lid off for 30 minutes, then replace lids and keep in 20oC incubator
20 °C
?. [Imaging (eg Wednesday)]
Repeat steps 17-25 for the next batch of worms for the next imaging run
?. [Imaging (eg Wednesday)]
For each imaging run, allow 30 minutes drying, 30 minutes in 20oC incubator with lids on and then 30 minutes acclimation in worm cave before imaging on hydra (calculate times from middle of wormsorter time)
?. [Imaging (eg Wednesday)]
Imaging on hydra using protocol script (5 mins prestim; 6 mins bluelight with 60 sec OFF, [10sec ON, 90sec OFF] x 3 times; 5 mins postsim)
~/scripts$ python run_syngenta_experiment_v2.py -f disease_models_run1 – r 01 02 03 04 05-f : project/file name for batch of recordings with run number at the end:! do not use blank spaces in file name!! add run number for each run of tracking on each day !-r : specifies which rigs to start recording on
?. [Transfer files from NAS]
Enter project/file name pattern to search for (eg disease_models; nb. all lower case)
?. [Transfer files from NAS]
Enter the folder where the files should be transferred
A new folder is automatically generated for each day of imaging