MattPiperLab: set up Drosophila lifespan experiment

materials

purps (egg laying plates)

• petri dishes
• grape juice
• agar

--> ensure sufficient agar in mix to yield a hard surface that won't be disturbed when washing off eggs

bottles

• half-pint milk bottles
• 70ml 1xSY food

(see Bass et al, 2007, J Gerontol, 62A,1071-81 for food making supplies and method)

1xSY food per litre:

• 15g agar (Sigma)
• 50g sucrose (Tate & Lyle)
• 100g lyophilised yeast (MPBIo brewer's yeast)
• 30ml nipagin solution (clariant UK Ltd) - (100 g/L methyl 4-hydroxybenzoate in 95% ethanol)
• 3ml propionic acid (Sigma)

Standard density fly rearing

Parental generation

• put purp in population cages overnight (for best results, purp cage twice over 48h and use the second purp for egg squirts)
• wash eggs from purps with PBS (dislodging with a paint brush) into falcon tube and allow to settle
• using a wide-bore tip (or cut off yellow tip 6mm from bottom) and a 100ul pipette, squirt eggs into 1.0xSY bottles (20ul per bottle should give about 250 – 300 flies per bottle)

(note that to uptake a densely-packed tip of eggs, rapidly release the plunger on the pipette and drop the tip further into the egg mass)

• on 10th day, transfer all flies that have emerged to fresh bottles and maintain tipping every-other-day until ready to set up cage for experimental generation

experimental generation

• put 4 to 5 bottles of parental flies into cage (1l yoghurt container cage)
• purp cages over 48h with live yeast paste, using the eggs from the overnight lay on the second purp for experimental flies
• wash eggs from purp with PBS into falcon tube and allow to settle
• cut off yellow tip and squirt 20ul eggs per bottle
• on the 9th day (evening) clear bottles
• on the 10th day transfer emerged flies to fresh bottles
• allow to mate for 2 days for once-mated females or male experiments.

Experimental set-up

anaesthetise flies, sex and count into vials or bottles of difft food concentrations. This is day 0 for experimental treatment.

anaesthetising flies

• put needle from gas outlet into a clean, dry bottle and allow to fill with CO2
• tip flies from bottle into this bottle of gas to knock-out, and transfer anaesthetised flies to CO2 pad
• while sexing / counting flies, put needle back in empty bottle to fill with gas again ready for next bottle of flies

allocating flies to treatments

• flies need to be allocated to different treatments systematically to avoid one bottle supplying all the flies to one or a few treatments:
• eg. When working with 5 food treatments (0.1, 0.5, 1.0, 1.5 and 2.0 SY) in vials

* working with anaesthetised flies from one bottle, transfer 10 flies to 0.1, then 0.5, then 1.0, then 1.5, then 2.0 * there should be enough flies from one bottle (~300) to do up to 3 vials per treatment in this case, if there aren’t enough remaining on the pad for another full set of treatments (ie 50 flies) discard remainders and start a new bottle * to control for duration of gassing, when working with the next bottle of anaesthetised flies, first allocate to treatment 0.5, then 1.0, then 1.5, then 2.0 and then 0.1.

example time-line

• Wed: put egg laying plate with live yeast paste into population cage overnight
• Thur: retrieve egg laying plate from population cage. late afternoon replace with a fresh plate with a small amount of live yeast paste
• Fri: in the morning, retrieve egg plate, wash off eggs to collect and perform egg squirts
• Sat:
• Sun:
• Mon:
• Tue:
• Wed:
• Thur:
• Fri:
• Sat:
• Sun:
• Mon: transfer freshly emerged flies into a new bottle (parental generation)
• Tue:
• Wed: gas flies and combine 3-4 bottles into cage with yeast paste on a fresh purp
• Thur: retrieve egg laying plate from population cage. late afternoon replace with a fresh plate with a small amount of live yeast paste
• Fri: in the morning, retrieve egg plate, wash off eggs to collect and perform egg squirts
• Sat:
• Sun:
• Mon:
• Tue:
• Wed:
• Thur:
• Fri:
• Sat:
• Sun:
• Mon: transfer freshly emerged flies into a new bottle
• Tue:
• Wed: gas flies and sort/allocate into vials for treatment (=day 0 of experiment)