Mar 04, 2025
Treating mast cells and basophils with degranulation inducers
- 1Arcadia Science
- Arcadia Science
Protocol Citation: Justin Donnelly 2025. Treating mast cells and basophils with degranulation inducers. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvjnezbgk5/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: 106287
Keywords: mast cell, basophil, degranulation, Compound 48/80, PMA, A23187, HMC1.2, RBL-2H3
Abstract
This protocol describes approaches to induce degranulation in RBL-2H3 (rat basophil) and HMC1.2 (human mast cell) lines. With appropriate positive and negative controls (described herein), the protocol can also be used to test whether a particular reagent induces or inhibits degranulation of these cells.
Materials
Calcium Ionophore A23187Merck MilliporeSigma (Sigma-Aldrich)Catalog #C7522 Dimethyl sulfoxide (DMSO)Merck MilliporeSigma (Sigma-Aldrich)Catalog #D2438 Phorbol 12-myristate 13-acetate (PMA)Merck MilliporeSigma (Sigma-Aldrich)Catalog #P8139 Compound 48/80Merck MilliporeSigma (Sigma-Aldrich)Catalog #C2313 Trypsin-EDTA (0.25%)Thermo Fisher ScientificCatalog #25200056
24-well plate
1.5 mL microcentrifuge tubes
Complete RBL-2H3 media
Complete HMC1.2 media
15 mL centrifuge tubes
Protocol materials
Calcium Ionophore A23187Merck MilliporeSigma (Sigma-Aldrich)Catalog #C7522
Dimethyl sulfoxide (DMSO)Merck MilliporeSigma (Sigma-Aldrich)Catalog #D2438
Phorbol 12-myristate 13-acetate (PMA)Merck MilliporeSigma (Sigma-Aldrich)Catalog #P8139
Compound 48/80Merck MilliporeSigma (Sigma-Aldrich)Catalog #C2313
Trypsin-EDTA (0.25%)Thermo Fisher ScientificCatalog #25200056
Trypsin-EDTA (0.25%)Thermo Fisher ScientificCatalog #25200056
Trypsin-EDTA (0.25%)Thermo Fisher ScientificCatalog #25200056
Compound 48/80Merck MilliporeSigma (Sigma-Aldrich)Catalog #C2313
Phorbol 12-myristate 13-acetate (PMA)Merck MilliporeSigma (Sigma-Aldrich)Catalog #P8139
Dimethyl sulfoxide (DMSO)Merck MilliporeSigma (Sigma-Aldrich)Catalog #D2438
1X PBS (Phosphate-buffered saline )
Compound 48/80Merck MilliporeSigma (Sigma-Aldrich)Catalog #C2313
Calcium Ionophore A23187Merck MilliporeSigma (Sigma-Aldrich)Catalog #C7522
Dimethyl sulfoxide (DMSO)Merck MilliporeSigma (Sigma-Aldrich)Catalog #D2438
1X PBS (Phosphate-buffered saline )
Safety warnings
For certain applications, it is necessary to use complete media lacking phenol red. Before performing an experiment, consider whether the presence of phenol red will interfere with your downstream application.
Inducing degranulation in RBL-2H3 cells
Inducing degranulation in RBL-2H3 cells
45m
45m
Plate 500,000 live RBL-2H3 cells per well in a 24-well plate.
Rinse the cell layer with Trypsin-EDTA (0.25%)Thermo Fisher ScientificCatalog #25200056 or PBS to remove traces of serum.
Add 2.0-3.0 mL of Trypsin-EDTA (0.25%)Thermo Fisher ScientificCatalog #25200056 solution to the flask. Incubate cells at 37 °C until they detach — for RBL-2H3, this is usually within 00:10:00 .
10m
Add 6.0-8.0 mL of complete growth medium (≥ 2× the trypsin volume) and dislodge remaining cells in the flask by gently pipetting.
Count cells. Prepare a mixture of 10 µL cells and 10 µL trypan blue, inverting cells gently before removing sample for counting. Mix well, then transfer 10 µL trypan-diluted cells to a Countess slide. Count using Countess to determine cell density and relative viability.
Prepare a cell suspension at a density of 1.0 million live cells/mL with a sufficient volume to plate in the desired number of wells. If cell density is < 1.0 million live cells/mL, centrifuge desired quantity of cells at 125 x g, 00:05:00 , then resuspend pellet in the appropriate volume of complete media.
5m
Transfer 0.5 mL cell suspension per well to desired quantity of wells in a 24-well plate.
Incubate cells Overnight .
The following day, prepare 1× treatment solutions in media without serum or phenol red.
Note
For certain applications, it's necessary to use complete media lacking serum and/or phenol red. For consistency, we always perform degranulation treatments in complete media without serum or phenol red, even when not strictly necessary.
For RBL-2H3, suitable positive controls include 30.0 µg/mL Compound 48/80Merck MilliporeSigma (Sigma-Aldrich)Catalog #C2313 or 10 nanomolar (nM) Phorbol 12-myristate 13-acetate (PMA)Merck MilliporeSigma (Sigma-Aldrich)Catalog #P8139 .
You should also prepare an appropriate negative control, such as Dimethyl sulfoxide (DMSO)Merck MilliporeSigma (Sigma-Aldrich)Catalog #D2438 or 1X PBS (Phosphate-buffered saline ) .
Note
If using DMSO as a negative control and degranulation agent stocks in DMSO, you must control the concentration of DMSO across samples for downstream applications, especially the hexosaminidase assay. DMSO absorbs light in the same wavelength region as the hexosaminidase assay fluorophore. We recommend using PBS-based stocks whenever possible.
Aspirate media from wells containing previously plated RBL-2H3. Wash cells with 0.5 mL PBS.
Aspirate PBS. Add 0.3 mL appropriate treatment solution to each well. Incubate cells for 00:30:00 .
30m
Using a pipette, collect supernatant for downstream analysis.
Inducing degranulation in HMC1.2 cells
Inducing degranulation in HMC1.2 cells
40m
40m
Prepare 2× treatment solutions in complete media.
Note
For certain applications, it's necessary to use complete media lacking phenol red. For consistency purposes, we always perform degranulation treatments in complete media without phenol red, even when not strictly necessary.
For HMC1.2, suitable positive controls include 30.0 µg/mL Compound 48/80Merck MilliporeSigma (Sigma-Aldrich)Catalog #C2313 or 100 nanomolar (nM) Calcium Ionophore A23187Merck MilliporeSigma (Sigma-Aldrich)Catalog #C7522 .
You should also prepare an appropriate negative control, such as Dimethyl sulfoxide (DMSO)Merck MilliporeSigma (Sigma-Aldrich)Catalog #D2438 or 1X PBS (Phosphate-buffered saline ) .
Note
If using DMSO as a negative control and degranulation agent stocks in DMSO, you must control the concentration of DMSO across samples for downstream applications, especially the hexosaminidase assay. DMSO absorbs light in the same wavelength region as the hexosaminidase assay fluorophore. We recommend using PBS-based stocks whenever possible.
Recover cells from culture flask. Dislodge any cells from the flask by gently pipetting.
Count cells. Prepare a mixture of 10 µL cells and 10 µL trypan blue, inverting cells gently before removing sample for counting. Mix well, then transfer 10 µL trypan-diluted cells to a Countess slide. Count using Countess to determine cell density and relative viability.
Prepare a cell suspension at a density of 6.0 million live cells/mL with a sufficient volume to plate in the desired number of wells. Centrifuge desired quantity of cells at 300 x g, 00:05:00 , then resuspend pellet in the appropriate volume of complete media.
5m
Transfer 150 µL cell suspension per well to desired quantity of wells in a 24-well plate.
Transfer 150 µL 2× treatment solution per well to appropriate wells. Incubate cells for
00:30:00 .
30m
After incubation, transfer well contents to clean 1.5 mL microcentrifuge tubes. Centrifuge at 300 x g, 00:05:00 . Collect supernatant for downstream analysis.
5m