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.
Adenosine aggravates pain via the interactions of adenosine A2A receptor (A2AR) and adenosine A1 receptor (A1R). And heteromerization of A2AR and A1R can put adenosine into operation. Simultaneously, a growing body of information indicates Ca2+ participate in pain management. Our team proved the interaction between A1R and A2AR by Iem-spFRET method. In the present study, we estimated the influence of the dimerization on the concentration of intracellular Ca2+. These dates demonstrate that the interaction of pain-related receptors (A2AR and A1R) has a significant effect on intracellular Ca2+ signaling.
Mast cells (MCs) degranulation have an extremely momentous role in the progresses of immunoreaction, anaphylaxis as well as the variation of the tumor microenvironment (TME). The emergence of the substances due to MCs degranulation will arouse multiple changes of optical characteristics, such as energy transfer, fluorescence and spectra, etc. In this study, we implement the simultaneous spectral unmixing of excitation and emission by adjusting the cube filters and optical path to solely trigger the donor excitation and obtain the acceptor fluorescence emission. In addition, we add another channel to collect the real-time spectra with a portable and mobile spectroscopy equipment. Here, we developed graphene oxide (GO) and reduced GO (rGO)-based fluorescence resonance energy transfer (FRET) biosensors for MCs degranulation to verify the performance of the dual-channel system on an Inverted Fluorescence Microscope. MCs undergo degranulation can rapidly release tryptase, one proteases of the highest concentration in cytozoic pre-formed mediator. The acceptor fluorescence emission and spectra are detected simultaneously in real-time by tryptase-sensitized FRET biosensor on the dual-channel system. Moreover, the dual-channel can be switched by rapid adjusting optical channel during excitation at any moment. Results showed that the MCs degranulation could be monitoring in real-time on the dual-channel optical system. In particular, the minimal changes of the initial degranulation also could be measured with high response rate. Consequently, this dual-channel system may serve as a potential tool for the investigation of protein-protein interaction, single molecule dynamics and the working mechanism of membrane proteins using FRET-related techniques.
During the development of tumors, some protein molecules, secreted proteins, are secreted, which are closely related to the proliferation, invasion and metastasis of malignant tumor cells. Therefore, the study of tumor cell secreted proteins not only helps to understand the molecular mechanism of tumorigenesis and development, but also helps to find new tumor markers for early screening of cancer and monitoring of high-risk populations. Surface-enhanced Raman spectroscopy (SERS) and partial least squares-support vector machine (PLS-SVM) data processing methods were used to characterize secreted proteins from human liver cancer cells HepG2 and normal human liver cells LO2 cells in this paper. The discriminative sensitivity and specificity of secreted proteins reach 100%, respectively. These results show that SERS technology combined with PLS-SVM data processing method can effectively distinguish normal cells from cancer cells and provide new ideas for finding biomarkers of cancer cells.
Independent emission-spectral unmixing fluorescence resonance energy transfer, Iem-spFRET, is a novel and powerful tool for measuring FRET efficiency in real time. In this paper, we simulate the measurement error of the Iem-spFRET by introducing random noise in sample data, donor fingerprint, and acceptor fingerprint. The random noise intensity is set from 0.0005 to 0.0025, corresponding to 5% - 25% of the maximum donor fingerprint intensity. The simulated results show the effect of random noise on apparent FRET efficiency (EfD) is less than on receptor-to-donor concentration ratio (Rc). Random noise with 10% maximum donor fingerprint intensity only leads to 0.33% variation of when the noise is added to both sample and fingerprints. These results indicate that Iem-spFRET is a robust method and could be applied on cases with weak FRET signal.
Degranulation in mast cell is usually characterized by the release of tryptase. We developed a fluorescence resonance energy transfer (FRET) probe based on graphene oxide (GO) to detect tryptase released from mast cells. The GO based FRET probe is composed of GO and a self-assembled complex of tryptase-specific recognition peptide chains labeled with isothiocyanate fluorescein. The fluorescence intensity around the mast cells increased when the mast cells were stimulated with C48/80, a kind of reagent promoting degranulation. The fluorescence distribution is inhomogenous. The fluorescence intensity was dependent on the concentration of C48/80 and the stimulation time. These results demonstrate that GO-base FRET probe could be used to study degranulation in mast cells.
Cancer cells secrete copious amounts of secretory granules, exosomes, proteases. Recently, studies reviewed that mast cells (MCs) play crucial roles in the growth, spread and metastasis of tumor. MCs are one of the earliest cell types to infiltrate developing tumors. MCs undergo degranulation in response to various stimuli and rapidly release diverse bioactive mediators, such as histamine, tryptase, serotonin, tumor necrosis factor α (TNFα), which will tremendously affect the tumor microenvironment (TME). However, the mechanisms between the secretion of MCs degranulation and tumor remain unclear. Therefore, we developed a nanobiosensor based on fluorescence resonance energy transfer (FRET) for the determination of P815 mast cells and HeLa cells by secretagogues. With the pep-FITC as an energy donor and reduced grapheme oxide (rGO) as an energy acceptor, the two parts assemble an efficient FRET biosensor through electrostatic and stacking interaction (- interaction). Sensitized secretory cells can produce tryptase which would hydrolyze the specific cleavage site of the peptide, leading to ruin FRET system and then yield intensive fluorescence (FL) recovery of quenched FITC. Results showed that P815 cells are more sensitive and intense secretory than HeLa cells owing to more amount secretory mediators of P815 can change the microenvironment and further exacerbate the degree of degranulation in return. Our findings suggest that FRET biosensor have the ability to detect the extracellular dynamics of the cancer cells microenvironment. In addition, targeting mast cells may serve as a novel therapeutic scheme for cancer treatment and that inhibiting mast cell function may lead to tumor regression.
Adenosine plays important roles in the pain signal transduction by activating adenosine receptors of two subtypes of A1 and A2A. In this study, FRET system based independent emission -spectral spectral spectral unmixing method (Iem-spFRET) was set up and used to measure the energy transfer from A1R to A2AR. The energy transfer efficiency calculated by Iem-spFRET is about 17.44%. All the above date and results demonstrate that FRET with special designed fluorescence proteins could be used to investigate the interaction between adenosine receptors.
In this study, two FRET-based probes are constructed to research oligomerization of Epstein-Barr virus Oncoprotein LMP1 in live cells. The images of wide-field fluorescence microscopy display that the majority of two LMP1-associated probes co-localized in internal perinuclear membranes. Furthermore, the fluorescence spectra of single cell co-expressed two probes indicated that the ratio of two emission peaks is around one, and the fluorescence spectra changed insignificantly during an hour observation. These findings indicated that LMP1/LMP1 interacted stably in live cells.
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