Single mast cell degranulation detection based on FRET probe

Mast cells (MCs) undergo degranulation activated by various secretagogues and rapidly secrete pre-formed mediators include in secretory granules, involving in numerous progresses of immune response, hypersensitivity and carcinogenesis. Therefore, it is essential for MCs degranulation detection in vivo and in real-time, particularly the measurement of MC degranulation at single cell level. At the aim of single cell degranulation detection, we here developed a secretion-sensitizing FRET probe, designated tryptase-sensitizing probe (Tryprobe). After treated with C48/80 or trypsin, activated MCs will degranulate to release inflammatory mediators. The release of tryptase in the process of degranulation will destroy the non-fluorescent FRET system of Tryprobe, and then the fluorescence production was detected by fluorescence spectrometer or CCD imaging equipment. Curcumin-pretreated P815 cells sensitized with C48/80, the fluorescence intensity of FRET system significantly declined than the drugs stimulation groups. From single cell scale, the cell morphology is nearly unchanged when cells under the rest state without drug activation. However, after drugs stimulation, the cell morphology is becoming distorting at various degrees, some cells morphology even break in intense degranulation. The morphology change of single mast cell can be observed distinctly in bright field and green fluorescent channel. To real-time observe single mast cell degranulation, we also established Intensity-Scatter correlation for the reconstruction of degranulation to build an evaluation standard for single cell degranulation using Tryprobe detection method. Accordingly, we anticipate this Intensity-Scatter correlation method using Tryprobe system as a template to be applied to other secretions detection of single cell in the cell and molecular biological fields.