According to protocols given by Sigma Aldrich

0.5 μl capillaries with adequate suction cup, 0.5ml and 1.5ml Eppendorf cap, ice, peaked stylus, 2 forceps (or scissors) to clip wings

Fly preparation:
Sting 3 holes in a 0.5ml Eppendorf cap (size of the holes is important: not too small so that enough haemolymph can go through, not too big so that no fat or other dirt is going through), put into 1.5ml Eppendorf cap with removed lid.
Remove the flies’ wings, spear the fly’s thorax with the peaked stylus
Collect 20 speared flies in the 0.5ml Eppendorf cap with holes, on ice
Centrifuge the 0.5ml Eppendorf cap within the 1.5ml one (1 min, 5000 rpm, at 4°C)
Discard the 0.5ml Eppendorf cap, soak the pellet with a capillary
Record the amount of soaked haemolymph (to fill up the 0.5μl you need around 50 flies)
The haemolymph from the capillary can be transferred with the suction cup anywhere

Enzymatic procedure:
add 19.5µl cold PBS to 0.5µl haemolymph
10µl of this mixture to 30µl Citrate Acid Buffer and 10µl of a 3% Trehalase-Citrate acid buffer solution
incubate over night at 37°C
add 50µl Tris Buffer
80µ of this mixture are added to 156.8µ Glucose oxidase (aliquot in the freezer) and 3.2µl o-Dianisidine (freshly added from the fridge)
incubate for exactly 30min at 37°C (timer!)
stop reaction by adding 160µl Sulfuric Acid
measure at 540nm at the (nanoDrop) spectrometer

Comments are welcome here or on figshare.

Print Friendly, PDF & Email