Mast Cell Degranulation Assay

Mast Cell Degranulation Assays

Mast cells, belonging to the myeloid lineage of immune cells, are distributed in connective tissues throughout the body. The activation and degranulation of mast cells play a significant role in modulating various physiological and pathological conditions.
In terms of normal physiological functions, mast cells are involved in regulating vasodilation, maintaining vascular homeostasis, mediating innate and adaptive immune responses, promoting angiogenesis, and facilitating venom detoxification.
Conversely, mast cells have also been implicated in the pathophysiology of numerous diseases such as allergy, asthma, anaphylaxis, gastrointestinal disorders, various malignancies, and cardiovascular diseases.
Mast cells express c-kit and FcεR1 receptors. Human mast cells can be classified into two phenotypes: mucosal mast cells that exclusively produce tryptase and connective tissue mast cells that produce chymase along with tryptase and carboxypeptidases.
The cytoplasm of a mast cell contains 50-200 large granules which store inflammatory mediators, i.e. histamine, heparin, a variety of cytokines, chondroitin sulfate, and neutral proteases.
Mast cell activation can occur through antigen/IgE/FcϵRI cross-linking as well as stem cell factor-induced c-kit dimerization.

OUR HUMAN MAST CELL ASSAYS WIDELY USED TO SYUDY BIOLOGY OF DISEASES

Challenges of Mast Cell Research

Primary human mast cells are rare in peripheral blood and tissue isolation protocols yield low cell quantities, viability and purity.
Differentiation of blood stem/precursor cells to mature mast cells is time-consuming and culture reagents are costly.
Many human (HMC-1, LAD2, LUVA) and rodent (P815, FMA-3) mast cell lines exhibit aberrant phenotypes, including lack/loss of functional receptors such as FcεR1, impaired cytokine production, long doubling time or insufficient cytosolic granules.

Primary Cells or Cell Line Availability: Ability to chose human CD34+ blood precursor-derived mast cell and/or RBL-2H3 cell line for compound testing. Additionally, both cell sources express functional receptors and cytosolic granules critical for functional studies.
Variety of readouts: histamine release assay/ tryptase/β-hexosaminidase/cytokines/PGD2/gene expression
Degranulation Induction: IgE-dependent and -independent (Compound 48/80, cortistatin-14, substance P)
High throughput: 96/384-well format, duplicate or triplicate; multiple donors can be tested concurrently; highly multiplex analysis at transcriptome/ secretome/proteome levels
Robust and highly reproducible: More predictive results than with cell lines or rodent cells
Well-validated reagents and protocol: provide established differentiation reagents, mast cell agonists and antagonists.

CD34+ precursor cell isolation from peripheral blood.
Differentiation of CD34+ precursor to mature mast cells
Evaluation of mast cell culture purity: c-kit and FcεR1 expression
Test compound treatment during stimulation
Readout quantification:
- Cytokines production
- Gene Expression
- Degranulation products/markers (histamine release assay, etc.)

Primary CD34+ hematopoietic precursor cells isolated from 2 healthy donors were differentiated to mast cells in vitro by culturing cells in medium supplemented with recombinant stem cell factor (SCF), interleukin (IL)-6, and IL-3. Mast cell culture purity was evaluated after weeks of differentiation based on the expression of c-kit and FcεR1 with flow cytometry.

Mature CD34+ hematopoietic precursor-derived mast cells were incubated with varying concentrations of test compound or cromolyn (mast cell stabilizer) during stimulation with compound 48/80 (mast cell activator). After treatment, β-hexosaminidase, histamine, prostaglandin D2 and tumor necrosis factor (TNF)-α in the cell-free supernatants were quantified. Data shown are mean ± SEM (3 donors).