In the past, electron microscopy was used to visualise and characterise mast cells.
Real-Time Imaging of Mast Cell Degranulation in Vitro and In Vivo - \citet*{Horiguchi_2016}
Developed a pH-sensitive system to visualise mast cell degranulation.
- conjugate a pH-sensitive green fluorescent protein (pHluorin) to Vesicle Associated Membrane Protein 8 (VAMP-8) at the C-terminal, creating a protein called immuno-pHluorin (impH). It only fluoresces at a neutral pH – when acidic secretory granules fuse with plasma membrane, pH increases due to exposure to neutral external environment.
- Transfect Bone marrow-derived mast cells (BMMCs) with retrovirus, transfectants sensitised with 2,4,6-trinitrophenol TNP-IgE, then activated with 300 ng/ml of TNP-conjugated ovalbumin (TNP-OVA) or control OVA.
- Transfected cells show no impairment in degranulation process measured by b-hexosamidase release assay. histamine release (shown by comparable histamine release between untranfested and impH mast cells treated with TNP-OVA after TNP-IgE stimulation). Figure 1 E
- Flow cytometric analysis showed transfection did not affect level of IgE receptors FcERI – FIgure 1D
- Functional evidence of impH via confocal microscopy:
- Increasing pH of transfected cells via NH4Cl and by blocking acidification with Bafilomycin A1 leads to presence of green fluorescent patches that are absent in PBS-treated cells.
- This pH switch is illustrated when sensitised cells were stimulated with TNP-OVA where impH-expressing cells showed green fluorescent patches near edge of cells within 10 minutes after treatment. Cells stimulated with control ova showed no green patches.
- The green patches co-localise with newly formed CD63-a degranulation marker-indicating that impH enables visualisation of mast cell degranulation in vitro.
- The impH system showed that the degranulation process is polarised –his is seen when they used a TNP-OVA conjugated beads with a 3 microm diamete. They proposed that it only occurs at certain sites where the antibody-allergen binds to and cross-links the FcERI (Figure 3). T To test this in vivo, --
- Able to show degranulation process in vivo using mast cell deficient mice reconstituted with either wild-type of FcRgamma-deficient BMMC. Inject the ear skin of mice interdermally with TNP-IgE prior to IV injection with TNP-OVA. Green patches only seen in mice reconstituted with wild-type BMMC, illustrating that the impH system utilises the engagement of the allergen bound-IgE receptors prior to degranulation.