Haemolymph extraction of adult Drosophila
Björn Brembs, Christine Damrau, Brembs' lab members
Abstract
This is a very simple protocol showing how to extract haemolymph from adult Drosophila melanogaster. (Based on protocols from Sigma Aldrich).
Before start
Steps
Fly preparation
Punch three holes in a 0.5ml Eppendorf cap and put it into a 1.5ml Eppendorf cap with removed lid.
Remove the flies’ wings, spear the fly’s thorax with a needly.
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).
0h 1m 0s
Discard the 0.5ml Eppendorf cap, collect the extracted hemolymph with a fine capillary.
Record the amount of haemolymph (to fill up 0.5μl you need around 50 flies).
Enzymatic procedure
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.
18h 0m 0s
Add 50µl Tris Buffer.
80µl 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.
0h 30m 0s
Stop reaction by adding 160µl Sulfuric Acid.
Measure at 540nm at the (nanoDrop) spectrometer.
