Mar 14, 2025

Public workspaceMaking Drosophila Artificial Hemolymph

DOI
dx.doi.org/10.17504/protocols.io.q26g7mqq9gwz/v1
  • 1University of Pittsburgh
Zachary Freyberg
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DOI: dx.doi.org/10.17504/protocols.io.q26g7mqq9gwz/v1
Protocol Citation: Zachary Freyberg 2025. Making Drosophila Artificial Hemolymph. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7mqq9gwz/v1
Manuscript citation:
Doi: 10.1038/ncomms10652
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: March 14, 2025
Last Modified: March 14, 2025
Protocol Integer ID: 124407
Keywords: Drosophila, Brain, Dissection,
Abstract
This is a protocol for making artificial hemolymph (AHL) for Drosophila brain dissection and imaging.
Materials
NaCl                 
KCl                        
CaCl2                      
MgCl2                     
NaH2PO4               
Sucrose          
Trehalose       
HEPES 
ddH2O
pH meter
Safety warnings
N/A
Ethics statement
N/A
Before start
Make sure to make the stock solutions ahead of making the final AHL buffer.
Making Drosophila Artificial Hemolymph (AHL)
Making Drosophila Artificial Hemolymph (AHL)
Make stock solutions
Stock Solutions
3M NaCl                       MW: 58.44       87.66g to 500mL 
1M KCl                         MW: 74.56       37.28g to 500mL 
500mM CaCl2                     MW: 147.00     14.7g to 200mL 
500mM MgCl2                     MW: 203.3       20.33g to 200mL
500mM NaH2PO4               MW: 155.99     15.59g to 200mL
*200mM Sucrose          MW: 342.30     34.23g to 500mL 
*100mM Trehalose       MW 378.33      3.78g to 100mL 
*250mM HEPES           MW: 238.31     11.92 g to 200mL 
*Store at 4°
Make 100 mL of AHL from the stock solutions
108mM NaCl                3.6mL  of 3M NaCl 
5mM KCl                     .5mL    of 1M KCl 
2mM CaCl2                            .4mL    of 500mM CaCl2
8.2mM MgCl2                      1.64mL of 500mM MgCl2
1mM NaH2PO4                    .2ml     of 500mM NaH2PO4
10mM Sucrose              5mL     of 200mM Sucrose
5mM Trehalose             5mL     of 100mM Trehalose 
5mM HEPES                2mL     of 250mM HEPES 
4mM NaHCO3              .4mL    of 1M NaHCO3
 
**add NaHCO3 last to avoid formation of precipitates
Add ddH2O to a final volume of 100 mL
Adjust final pH to 7.5
Check osmolarity of the solution to ensure that it is at ~265 mOsm
Protocol references
Freyberg, Z, Sonders, MS, Aguilar, JI, Hiranita, T, Karam, CS, Flores, J, Pizzo, AB, Zhang, Y, Farino, ZJ, Chen, A, Martin, CA, Kopajtic, TA, Fei, H, Hu, G, Lin, YY, Mosharov, EV, McCabe, BD, Freyberg, R, Wimalasena, K, Hsin, LW, Sames, D, Krantz, DE, Katz, JL, Sulzer, D & Javitch, JA 2016, 'Mechanisms of amphetamine action illuminated through optical monitoring of dopamine synaptic vesicles in Drosophila brain', Nature communications, vol. 7, 10652. https://doi.org/10.1038/ncomms10652