TITLE: Drug Behaviour Imaging on Phenix
AUTHORS: Ida Barlow, Adam Mcdermott-Rouse

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">For imaging drug-treated young adult C. elegans in liquids using the Multiworm tracker. Worms are synchronised by picking L4s, and then the young adults are exposed to drugs for 4 hours prior to imaging for 15 mins in a liquid droplet on a coverslip mounted on a 3.5cm plate.</div></div>

[STEPS]
?. [Making up the drugs (-1 Days)]
First identify the drugs to be used in the study and ensure that they are correctly labelled and handled.
?. [Making up the drugs (-1 Days)]
Calculate the desired weight and volume of solvent required for the desired concentration if using powdered compounds. Alternatively calculate the desired dilution for liquid compounds.
Important point – Ensure that your calculations are 1000 fold higher at this stage as your compound will be diluted when it is added to the plate. E.g. mM for uM active concentration
?. [Making up the drugs (-1 Days)]
Inside a fume hood with an analytical balance weigh out the desired amount of compound into an Eppendorf tube labelled with the compound and the final concentration.
?. [Making up the drugs (-1 Days)]
While still in the fume hood add the desired amount of solvent (eg DMSO) to get the right compound concentration, and close the lid of the Eppendorf tube
?. [Making up the drugs (-1 Days)]
Finally, to ensure the compound is dissolved fully, vortex the Eppendorf tubes (ensuring the lid is firmly in place) at setting 10 for 30 seconds.
NOTE: Some compounds require longer vortexing times (especially at higher concentrations) to ensure they completely dissolve. In these instances, repeat until fully dissolved.
?. [Making drug plates (-1 Days) ]
Select the appropriate number of fresh unseeded 35mm NGM plates (no older than 3 days) and inspect to ensure an even level of agar.
NOTES: If plates are uneven the images can appear blurry as the different height can result in out of focus patched.
Different level of agar indicates that there is a different volume and so the resultant concentration of drug will be affected
?. [Making drug plates (-1 Days) ]
Label the side of each plate with the compound name, compound concentration and the date.See the attached spreadsheet as a template for planning the imaging throughout the day
?. [Making drug plates (-1 Days) ]
Inside a flow hood apply 50ul of sterile water to the centre of the agar (this is to help the drug spread over the agar surface where the OP50 will be applied)
?. [Making drug plates (-1 Days) ]
Before this dries apply 3.5ul of your compound solution (ensuring the correct compound and concentration is used).
?. [Making drug plates (-1 Days) ]
Allow to dry in the flow hood with the lids off (this should take around 30 minutes).
?. [Making drug plates (-1 Days) ]
Once dry seed the plates with 50ul OP50 into the centre of each drug plate from the fridge
NOTES:Take a falcon tube of OP50 from the fridge and vortex on setting 10 until all the pellet has dissolved and not visible clumps remain in the solution.Dilute the OP50 1:10 in M9 solutionNormally when doing this stage, you might have many plates to do, in these instances the ones that were drugged first will probably dry before you have finished. Therefore, check for plates that have dried and replace the lids to ensure that there is no overdrying.
?. [Making drug plates (-1 Days) ]
Leave the plates overnight at room temperature in the dark.
?. [Drug exposure (Day 0, AM)]
First identify and label plates in accordance with the experimental Excel template.
?. [Drug exposure (Day 0, AM)]
For each set pick 10 worms from your synchronised plates onto each of the drug plates. This should be completed within 10 minutes for each set to ensure equivalence.
?. [Drug exposure (Day 0, AM)]
After 3h 30m has elapsed transfer 10 worms to the drugged imaging platesLeave to accimate o the new plate for 30 mins
?. [Imaging (Day 0, PM)]
Image for 15 minutes at 25fps with the .hdf5 file format.
?. [Imaging (Day 0, PM)]
Transfer the files to the server.
?. [Preparing wroms (-5 days)]
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?. [Preparing Behaviour plates (-3 day)]
{"blocks":[{"key":"37sqq","text":"C. elegans is maintained in the laboratory on Nematode Growth Medium (NGM) agar which has been aseptically poured into petri plates. The NGM agar medium can be poured into petri plates easily and aseptically using a peristaltic pump. This pump can be adjusted so that a constant amount of NGM agar is dispensed into each petri plate. A constant amount of agar in the plates reduces the need for refocusing the microscope when you switch from one plate to another.","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]},{"key":"7a41p","text":"","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]},{"key":"70s5t","text":"For imaging purposes, low peptone NGM is prepared which contains less concentration of bactopeptone as compared to the normal NGM. This is because we don't want the seeded bacteria to grow too much and obscure the images, rather just sufficient enough to keep the worms from crawling out of the plate due to lack of food. The imaging plates can be 35mm, 60mm or 90mm in diameter depending on the assay design.","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]},{"key":"fo0e9","text":"","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]},{"key":"27lv3","text":"","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]},{"key":"644a","text":"","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]}],"entityMap":[]}
?. [Refeed worms (-2.5 day)]
Refeed L1s to get young adults on the day of tracking