Mar 04, 2025

Public workspaceDetecting degranulation via hexosaminidase assay

DOI
dx.doi.org/10.17504/protocols.io.j8nlk8y65l5r/v1
  • 1Arcadia Science
  • Arcadia Science
Arcadia Science
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DOI: dx.doi.org/10.17504/protocols.io.j8nlk8y65l5r/v1
Protocol Citation: Justin Donnelly 2025. Detecting degranulation via hexosaminidase assay. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlk8y65l5r/v1
Manuscript citation:
Borges AL, Donnelly J, Morazan E, Rollins M. (2025). Compound 48/80 is toxic in HMC1.2 and RBL-2H3 cells. https://doi.org/10.57844/arcadia-3207-4695
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: August 22, 2024
Last Modified: March 13, 2025
Protocol Integer ID: 106297
Keywords: mast cell, basophil, degranulation, hexosaminidase, enzyme activity assay
Abstract
This protocol describes the quantification of hexosaminidase in cell culture supernatants via an enzyme activity assay. You can use hexosaminidase levels to determine whether mast cells or basophils have undergone degranulation. This protocol uses the Cell BioLabs beta-hexosaminidase assay kit (Fisher Scientific (NC1634727).

Materials
ReagentBeta-Hexosaminidase Assay KitFisher ScientificCatalog #NC1634727
Black 96-well microtiter plate
Plate reader
1.5 mL microcentrifuge tubes
15 mL centrifuge tubes
Cell culture supernatants (analyte)
Protocol materials
ReagentBeta-Hexosaminidase Assay KitFisher ScientificCatalog #NC1634727
Materials, Step 1
Reagent preparation
Reagent preparation
This protocol uses the ReagentBeta-Hexosaminidase Assay KitFisher ScientificCatalog #NC1634727 . If you've already prepared kit reagents, skip to "Standard and substrate preparation."

Dilute 5× assay buffer to 1× with ultrapure water. Vortex to homogeneity. Store at TemperatureRoom temperature .

Dilute 10× neutralization buffer to 1× with ultrapure water. Vortex to homogeneity. Store at TemperatureRoom temperature .

Standard and substrate preparation
Standard and substrate preparation
Thaw hexosaminidase positive control and 10× substrate TemperatureOn ice .

Just prior to use, prepare Amount35 µL positive control at Concentration5 µg/mL (10-fold dilution) in 1× assay buffer.

Prepare control solutions as follows:
ABCD
ControlVolume 5 µg/mL hexosaminidase (µL)Volume buffer (µL)Volume per well (µL)
500 ng/mL hexosaminidase17.5157.550
250 ng/mL hexosaminidase8.75166.2550
125 ng/mL hexosaminidase4.38170.6250
0 ng/mL hexosaminidase017550
Blank0350100

Dilute 10× substrate to 1× in assay buffer. Vortex to mix. Prepare only enough 1× substrate for immediate use (Amount50 µL per well, not including blanks).

Sample preparation and analysis
Sample preparation and analysis
15m
15m
Perform assay in technical triplicate.

Add Amount50 µL of each sample or control per well to appropriate wells of a black, 96-well microtiter plate (except blank wells, which get Amount100 µL ).

Add Amount50 µL 1× substrate per well to all wells except the blank.

Incubate at Temperature37 °C for Duration00:15:00 , protected from light.

15m
Add Amount100 µL of 1× neutralization buffer to each well.

Read each well's fluorescence at an excitation wavelength of 365 nm and an emission wavelength of 450 nm.
Note
DMSO absorbs significantly at 365 nm. If using DMSO, ensure that its concentration is normalized across all samples.

Protocol references