Mar 05, 2025
Detecting degranulation via hexosaminidase assay
- Justin Donnelly1
- 1Arcadia Science
- Arcadia Science
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 23, 2024
Last Modified: March 14, 2025
Protocol Integer ID: 106297
Keywords: mast cell, basophil, degranulation, hexosaminidase, enzyme activity assay, hexosaminidase assay kit, mast cell, degranulation via hexosaminidase, quantification of hexosaminidase, hexosaminidase level, cell culture supernatants via an enzyme activity assay, detecting degranulation, enzyme activity assay, hexosaminidase, cell culture supernatant, basophil, undergone degranulation, cell culture, assay, cell biolabs beta, degranulation
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
Beta-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
Beta-Hexosaminidase Assay KitFisher ScientificCatalog #NC1634727
Troubleshooting
Reagent preparation
This protocol uses the Beta-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 Room temperature .
Dilute 10× neutralization buffer to 1× with ultrapure water. Vortex to homogeneity. Store at Room temperature .
Standard and substrate preparation
Thaw hexosaminidase positive control and 10× substrate On ice .
Just prior to use, prepare 35 µL positive control at 5 µg/mL (10-fold dilution) in 1× assay buffer.
Prepare control solutions as follows:
| A | B | C | D | |
| Control | Volume 5 µg/mL hexosaminidase (µL) | Volume buffer (µL) | Volume per well (µL) | |
| 500 ng/mL hexosaminidase | 17.5 | 157.5 | 50 | |
| 250 ng/mL hexosaminidase | 8.75 | 166.25 | 50 | |
| 125 ng/mL hexosaminidase | 4.38 | 170.62 | 50 | |
| 0 ng/mL hexosaminidase | 0 | 175 | 50 | |
| Blank | 0 | 350 | 100 |
Dilute 10× substrate to 1× in assay buffer. Vortex to mix. Prepare only enough 1× substrate for immediate use (50 µL per well, not including blanks).
Sample preparation and analysis
15m
Perform assay in technical triplicate.
Add 50 µL of each sample or control per well to appropriate wells of a black, 96-well microtiter plate (except blank wells, which get 100 µL ).
Add 50 µL 1× substrate per well to all wells except the blank.
Incubate at 37 °C for 00:15:00 , protected from light.
15m
Add 100 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