Haemolymph extraction of adult Drosophila
Sting 3 holes in a 0.5ml Eppendorf cap and put into 15ml 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.
Add 19.5µl cold PBS to 0.5µl haemolymph.
Add 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 30 min at 37°C.
Stop reaction by adding 160µl Sulfuric Acid.
Measure at 540nm at the (nanoDrop) spectrometer.
