Apr 17, 2026
Protocol for Assessing Lysozyme-Like Activity in Galleria mellonella Hemolymph using EnzChek Kit
- Juan Jose Quispe Haro1,
- Adella Josephin2
- 1University of Helsinki, Faculty of Pharmacy;
- 2University of Helsinki, Faculty of Agriculture and Forestry
- Galleria protocols
Protocol Citation: Juan Jose Quispe Haro, Adella Josephin 2026. Protocol for Assessing Lysozyme-Like Activity in Galleria mellonella Hemolymph using EnzChek Kit. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gp7kw1gzp/v1
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: April 16, 2026
Last Modified: April 17, 2026
Protocol Integer ID: 315161
Keywords: Galleria mellonela, hemolymph, anticoagulant, phenoloxidase, infection, phenoloxidase activity in galleria mellonella hemolymph, free hemolymph of galleria mellonella larvae, like activity in galleria mellonella hemolymph, assessing lysozyme, galleria mellonella hemolymph, fungal infection in galleria mellonella larvae, assessing phenoloxidase activity, galleria mellonella larvae, lysozyme, use the same hemolymph sample, same hemolymph sample, antimicrobial enzymatic activity as the body, antimicrobial enzymatic activity, immune response in the host, free hemolymph, enzymatic activity, anticoagulant buffer, like enzymatic activity in the cell, fungal infection, fungal strain, immune response, like enzymatic activity, enzchek lysozyme kit, robust reading of lysozyme, using enzchek kit bacterial, enzchek kit bacterial
Disclaimer
DISCLAIMER – FOR INFORMATIONAL PURPOSES ONLY; USE AT YOUR OWN RISK
The protocol content here is for informational purposes only and does not constitute legal, medical, clinical, or safety advice, or otherwise; content added to protocols.io is not peer reviewed and may not have undergone a formal approval of any kind. Information presented in this protocol should not substitute for independent professional judgment, advice, diagnosis, or treatment. Any action you take or refrain from taking using or relying upon the information presented here is strictly at your own risk. You agree that neither the Company nor any of the authors, contributors, administrators, or anyone else associated with protocols.io, can be held responsible for your use of the information contained in or linked to this protocol or any of our Sites/Apps and Services.
Abstract
Bacterial or fungal infection in Galleria mellonella larvae produces an immune response in the host, affecting the antimicrobial enzymatic activity as the body tries to fight back the foreign elements causing the infection.
This is a protocol to evaluate lysozyme-like enzymatic activity in the cell-free hemolymph of Galleria mellonella larvae previously infected with a bacterial or fungal strain.
This is a fork from the previous protocol, modified to use the EnzChek Lysozyme Kit, for a more robust reading of lysozyme-like activity.
The anticoagulant buffer is taken from the described in DOI: 10.1159/000168009
Guidelines
Components of the EnzCheck Kit are light sensitive so prepare the reagents in the dark or a red lamp to avoid damaging the substrate
Materials
Anticoagulant Solution:
93 mM NaCl, 100 mM glucose, 30 mM trisodium citrate, 26 mM citric acid, 10 mM Na2EDTA, and 0.1 mM phenylthiourea, pH 4.6. Filter-sterilized through a 0.45 nm pore filter
Sterile PBS
EnzCheck Lysozyme Assay Kit
1.5 ml Eppendorf tubes
Crushed ice tray
Liquid nitrogen for snap-freezing
Refrigerated ultracentrifuge
0.2 ml microtubes
Dark plastic 384 well plates
Fluorescence microplate reader
Safety warnings
Ensure all equipment and reagents are sterile to prevent contamination.
It is crucial to keep all samples on ice until they are processed to maintain enzyme activity.
Ethics statement
Ensure to follow all safety and ethical guidelines when handling live larvae and biological materials.
Before start
Prepare anticoagulant buffer 93 mM NaCl, 100 mM glucose, 30 mM trisodium citrate, 26 mM citric acid, 10 mM Na2EDTA, and 0.1 mM phenylthiourea, pH 4.6
The day before
Grow 3 ml of an overnight bacteria culture at 37 °C in an incubator with vigorous shaking (150-200 rpm)
Bacteria preparation
10m
Take the overnight bacterial culture and transfer 2 ml into an Eppendorf tube
Centrifuge at no more that 1000 rcf, for 10 min at 4 °C.
10m
Discard the supernatant and resuspend the bacterial pellet in 1 ml of sterile PBS
Measure the absorbance at 600 nm and dilute the suspension so it contains about 10^6 cells/ml
Larvae injection
6h
Select Galleria mellonela larvae in their 7th instar (usually, above 400 mg in weight) and distribute in Petri dishes as groups of 5 for each condition.
Prepare a Hamilton syringe with 10 µl of bacterial suspension or sterile PBS in case of control.
Grab a larva by its sides with the thumb and the index fingers and flip it upside down to expose its abdomen
Localize the hind/left proleg of the larva and carefully insert the needle of the syringe about 5 mm into the body of the larva
Inject the contents of the syringe slowly to prevent any leaking, and remove the syringe.
Return the larva to their respective dish and repeat for all the larvae and conditions.
Transfer the Petri dishes to an incubator and incubate the larvae for 0-6 hours at 37 °C
6h
Material Preparation
2m
Dispense 30 µl of anticoagulant buffer into 0.2 ml microfuge tubes, and register their respective mass (1 tube per larva).
Transfer the tubes to an ice tray and wait a couple of minutes so the contents of the tubes are ice-cold.
2m
Hemolymph Collection
10m
Optional: Anesthetize the larvae on ice for 5-10 minutes to minimize movement during hemolymph collection
10m
Surface sterilize the larvae by spraying them with 70% ethanol over a paper towel and allow them to air dry.
Surface sterilize the blades of a pair of micro scissors or a scalpel by spraying them with 70% ethanol and allowing them to air dry.
With the blade, make a small cut in a abdomen of the larva.
Gently squeeze the larva to collect the dripping hemolymph (aim for 30-100 µl per larva) into a pre-chilled tube containing 30 µl of ice-cold anticoagulant buffer.
Optional: use 100 µl pipette to collect the last fraction of hemolymph
Place the tube back on ice immediately after collection.
Repeat the collection steps for all the larvae.
Optional: Flash freeze the tubes in liquid nitrogen and store at -20 °C until needed.
Preparation of Cell-Free Hemolymph (CFH)
1m
Register the weight and add ice cold anticoagulant buffer to ensure the final ratio of hemolymph to buffer is 1:1
Centrifuge the tubes at table top centrifuge at 12000 rpm for 1 minute to separate cellular debris from the hemolymph.
Optional: If centrifuging at 4 °C, you can use 4000 x g for 10 minutes.
1m
Optional: Carefully transfer aliquots of the clear supernatant (cell-free hemolymph) to a new pre-chilled tube.
Optional: Snap-freeze aliquots in liquid nitrogen and store at -20°C until ready for the next assay.
EnzChek Kit Standard curve
35m
The following steps are done in the dark to avoid the formation of fluorescein conjugates.
Prepare 8 microtubes with 50 μl of 1x reaction buffer
Add 50 μl of 1000 U/ml stock of lysozyme to the first tube. Mix well by pippetting
Prepare 1:2 serial dilutions with the next 6 tubes tube. The last tube should remain with reaction buffer only.
Add 50 μl of 50 μg/ml DQ lysozyme substrate to all 8 tubes and mix.
In a black plastic 384 well plate, add 30 μl of each mixture to three consecutive wells.
5m
Cover the plate with a transparent lid or seal film, and then wrap in aluminum foil and incubate at 37 °C for 30 minutes.
Measure the fluorescence intensity with a top-reading microplate reader.
Ex: 489 nm. Em: 523 nm
Optional: Measure every 5 minutes for 30 minutes total to get an average read value.
EnzChek Lysozyme-like assay
All steps are done in the dark.
Mix 25 μl of CFH with 25 μl of 1x reaction buffer.
Pipette 15 μl of this mixture into three consecutive wells of a black plastic 384-well plate.
Add 15 μl of 50 μg/ml DQ lysozyme substrate to each well.
Cover the plate with a transparent lid or seal film, and then wrap in aluminum foil and incubate at 37 °C for 30 minutes.
Measure the fluorescence intensity with a top-reading microplate reader.
Ex: 489 nm. Em: 523 nm
Optional: Measure every 5 minutes for 30 minutes total to get an average read value.
Data analysis
Generate the standard curve by averaging the fluorescence readings per concentration, and performing a linear regression.
Pool the readings of each sample set and average the fluorescence intensity reading.
Use this value to calculate the lysozyme activity equivalent of the samples from the standard curve.
Protocol references
Bidla, Gawa, Thomas Hauling, Mitchell S. Dushay, and Ulrich Theopold. "Activation of insect phenoloxidase after injury: endogenous versus foreign elicitors." Journal of Innate Immunity 1, no. 4 (2009): 301-308.
EnzChek Protocol https://documents.thermofisher.com/TFS-Assets/LSG/manuals/mp22013.pdf Accessed on 16.04.2026