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 .
