Synapto-phluorin Transfection
Synapto-phluorin Transfection - Related Videos
09:41
Confocal Imaging of Neuropeptide Y-pHluorin: A Technique to Visualize Insulin Granule Exocytosis in Intact Murine and Human Islets
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Cited by 12 •
2017
We describe a protocol for visualization of insulin exocytosis in intact islets using pHluorin, a pH-sensitive green fluorescent protein. Isolated islets are infected with adenovirus encoding pHluorin coupled to the vesicle cargo neuropeptide Y. This allows for the detection of insulin granule fusion events by confocal microscopy.
07:28
An Introduction to Transfection
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2023
Transfection is the process of inserting genetic material, such as DNA and double stranded RNA, into mammalian cells. The insertion of DNA into a cell enables the expression, or production, of proteins using the cells own machinery, whereas insertion of RNA into a cell is used to down-regulate the production of a specific protein by stopping translation. While the site of action for transfected RNA is the cytoplasm, DNA must be transported to the nucleus for effective transfection. There, the...
08:04
Neuronal Transfection Methods
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2023
Transfection - the process of transferring genetic material into cells - is a powerful tool for the rapid and efficient manipulation of gene expression in cells. Because this method can be used to silence the expression of specific proteins or to drive the expression of foreign or modified proteins, transfection is an extremely useful tool in the study of the cellular and molecular processes that govern neuron function. However, mature neurons have a number of properties that make them...
09:59
Utilizing pHluorin-tagged Receptors to Monitor Subcellular Localization and Trafficking
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Cited by 3 •
2017
Labeling the extracellular domain of a membrane protein with a pH sensitive fluorophore, superecliptic pHluorin (SEP), allows subcellular localization, expression, and trafficking to be determined. Imaging SEP-labeled proteins with total internal reflection fluorescence microscopy (TIRFM) enables the quantification of protein levels in the peripheral ER and plasma membrane.
04:21
Protein Transfection of Mouse Lung
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Cited by 1 •
2013
Transgenic mice or viral vectors have been used to increase protein expression within the lung. However, these techniques are time-consuming, technically challenging and have off-target effects that can confound results. Our protein transfection protocol uses a lipid based transfection reagent and an ultrafine microsprayer to uniformly deliver active protein to lung cells.
FAQs
pHluorin responds to the chemical environment surrounding the reporter. While a synaptic vesicle remains inside the neuron, its acidic lumen suppresses fluorescence. Fusion exposes the reporter to the neutral extracellular environment, increasing the signal. After endocytosis, reacidification lowers fluorescence again, allowing changes in intensity to follow successive stages of vesicle cycling.
The vesicle-associated protein directs the pH-sensitive reporter to synaptic vesicles, placing pHluorin where vesicle cycling occurs. This localization connects a fluorescence change to presynaptic membrane traffic rather than to general fluorescence throughout the neuron. As a result, the reporter can reveal activity-linked changes at synaptic sites and support analysis of presynaptic function.
A rise in fluorescence indicates that vesicle fusion has exposed pHluorin to the neutral extracellular environment, providing a readout of exocytosis. The subsequent decrease follows endocytosis and reacidification, which return the reporter to a less fluorescent state. Tracking these opposing signal changes over time therefore links presynaptic activity with release and recycling dynamics.
Researchers introduce the genetically encoded reporter into neurons, allow the construct to associate with synaptic vesicles, and observe fluorescence while the living cells experience neuronal activity. Changes in signal are then related to vesicle fusion and recovery. This workflow provides a way to examine synaptic behavior dynamically rather than relying only on a fixed endpoint.
Fluorescence recordings can reveal activity-linked release dynamics and the sequence of vesicle exocytosis, endocytosis, and recycling. Researchers can use these signals to assess how presynaptic function changes between experimental conditions or across neuronal preparations. The resulting comparisons help identify differences in synaptic transmission without treating fluorescence as a general measure of neuronal activity.
This approach is useful when investigators need to compare synaptic vesicle behavior in living neurons under different biological or experimental conditions. It can support studies of synaptic development, disease-related changes, and treatment effects by showing how release dynamics or recycling responses are altered. The method therefore connects molecular reporter signals with functional changes in synaptic transmission.