Jun 10, 2022

Public workspaceHaemolymph extraction of adult Drosophila V.3

DOI
dx.doi.org/10.17504/protocols.io.eq2lyd5elx9k/v3
  • 1Universität Regensburg;
  • 2Björn Brembs lab, Universität Regensburg
Björn Brembs
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DOI: dx.doi.org/10.17504/protocols.io.eq2lyd5elx9k/v3
External link: https://doi.org/10.3389/fnsys.2017.00100
Protocol Citation: Björn Brembs, Christine Damrau, Brembs' lab members 2022. Haemolymph extraction of adult Drosophila. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2lyd5elx9k/v3Version created by Björn Brembs
Manuscript citation:
Damrau C, Toshima N, Tanimura T, Brembs B, Colomb J, Octopamine and Tyramine Contribute Separately to the Counter-Regulatory Response to Sugar Deficit in . Frontiers in Systems Neuroscience doi: 10.3389/fnsys.2017.00100
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: June 10, 2022
Last Modified: June 10, 2022
Protocol Integer ID: 64321
Abstract
This is a very simple protocol showing how to extract haemolymph from adult Drosophila melanogaster. (Based on protocols from Sigma Aldrich).
Fly preparation
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).
Duration00:01:00
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
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.
Duration18:00:00
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.
Duration00:30:00
Stop reaction by adding 160µl Sulfuric Acid.
Measure at 540nm at the (nanoDrop) spectrometer.