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1. Measuring NPY-mRFP Exocytosis
<ol>
	<li>Preparation of Tyrode buffer:
	<ol>
		<li>Prepare a solution of 54 mM KCl, 20 mM MgCl2, 2.74 M NaCl, and 8 mM NaH2PO4 in DDW. Mix well and store at 4 &#176;C. This step is for the preparation of 20x Tyrode buffer.</li>
		<li>Prepare a solution of 20 mM Hepes pH 7, 1.8 mM CaCl2, 1 mg/ml BSA, 5.6 mM glucose, and 1 to 20 dilutions of Tyrode 20x in DDW and mix well. This step is for the preparation of 1x Tyrode buffer.</li>
		<li>Aliquot the 1x Tyrode buffer and store it at -20 &#176;C. Avoid repeated freezing and thawing.</li>
	</ol>
	</li>
	<li>Remove the culture medium from the 24 well plates containing the mucosal mast cell line, rat basophilic leukemia (RBL)-2H3 transfected with Neuropeptide Y fused monomeric RFP ( NPYmRFP) secretory granules reporter gene and wash 3 times with Tyrode buffer. Prepare unstimulated cells by adding 200 &#956;l Tyrode buffer to control wells.</li>
	<li>Prepare 10 &#956;l/well of 20X concentrated activating reagent [(e.g. 1,000 ng/ml DNP-BSA or DNP-HSA (Ag), 200 &#181;M Ca2+ionophore (e.g. A23187), and a combination of 20 &#181;M Ca2+ ionophore and 1,000 nM 12-O-&#160; tetradecanoylphorbol-13-acetate (TPA)]. If the reagents are stored in DMSO, dilute the reagents into 20x concentration in Tyrode buffer containing 1% DMSO. Incubate at 37 &#176;C for 30 min.</li>
	<li>Remove the supernatants of each well carefully to a 96-well plate, place on ice, and avoid light. (The supernatants contain the chimeric peptide NPY-mRFP that was released from the cells).</li>
	<li>Add 200 &#956;l Tyrode buffer containing 0.5% Triton X-100 to each well and incubate at 37 &#176;C for 10 min. (This step is important for the preparation of cell lysates that contain the remaining NPY-mRFP that was not released from the cells). Collect the cell lysates and transfer them to a 96-well plate, place them on ice, and avoid light.</li>
	<li>Measure the fluorescence of the cell supernatants and cell lysates using a fluorescence plate reader, using a 590-, 20 nm bandwidth excitation filter and 635-, 35 nm bandwidth emission filter.</li>
	<li>Calculate the percentage of NPY-mRFP released: 
	NOTE: The fluorescence reader measures arbitrary fluorescence units (AFU). AFU values depend on the machine, its sensitivity, and the transfection efficiency.
	<ol>
		<li>Set the autofluorescence of nontransfected RBL cells as blank. Divide the AFU of each supernatant by the total fluorescence (AFU of the supernatants + AFU of the corresponding lysate) and multiply by 100.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>DMEM</td>
			<td>Sigma-Aldrich</td>
			<td>D6046-500ML</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>GE health care Life sciences</td>
			<td>SH30071.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Life technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cellulose acetate membrane, pore size 0.22 &#956;m</td>
			<td>Sigma-Aldrich</td>
			<td>CLS430769-1EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning tissue-culture treated culture dishes</td>
			<td>Sigma-Aldrich</td>
			<td>CLS430167</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin/EDTA Solution (TE)</td>
			<td>Life technologies</td>
			<td>R001100</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr>
			<td>24 well, flat bottom</td>
			<td>Sigma-Aldrich</td>
			<td>CLS3524</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning 96-well plates</td>
			<td>Sigma-Aldrich</td>
			<td>CLS3367 or CLS390</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate fluorescence readerInfinite 200</td>
			<td>Tecan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Triton-X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787</td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring mast cell exocytosis via neuropeptide y monomeric red fluorescent protein release

Source: Azouz, N. P., et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/52505">Investigating Mast Cell Secretory Granules; from Biosynthesis to Exocytosis.</a> J. Vis. Exp. (2015)
This video demonstrates mast cell exocytosis induced by an activating reagent that includes a calcium ionophore, initiating the release of secretory granules containing the fluorescent reporter protein NPY-mRFP. Subsequently, we quantitatively assess the exocytosis by measuring the released NPY-mRFP using a fluorescence plate reader.

Measuring Mast Cell Exocytosis via Neuropeptide Y Monomeric Red Fluorescent Protein Release

1. Measuring NPY-mRFP Exocytosis

	Preparation of Tyrode buffer:
	
		Prepare a solution of 54 mM KCl, 20 mM MgCl2, 2.74 M NaCl, and 8 mM NaH2PO4 in DDW. ...

Take transfected mast cells expressing NPY-mRFP, a fusion protein containing neuropeptide Y and monomeric red fluorescent protein, in their cytoplasmic secretory granules.
Remove the media. Pipette calcium-containing buffer with an activating reagent, a calcium ionophore, and incubate.
These ionophores induce calcium influx, increasing the intracellular calcium concentration and mobilizing secretory granules to the plasma membrane.
The calcium binds to calcium-binding proteins on the secretory granule membrane.
Further, the secretory granules fuse with the plasma membrane mediated by membrane-bound proteins, resulting in exocytosis and release of contents, including NPY-mRFP.
Post-incubation, collect and transfer the released NPY-mRFP-containing supernatant to a black multi-well plate.
Add a non-ionic detergent to lyse the cells and release the remaining intracellular NPY-mRFP.
Collect and transfer the cell lysate to the multi-well plate.
Using a fluorescence plate reader, measure the fluorescence of the cell supernatant and lysate to quantify the percentage of NPY-mRFP released and determine the extent of mast cell exocytosis.

measuring-mast-cell-exocytosis-via-neuropeptide-y-monomeric-red

Nanyang Technological University

Research

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Cellular Immunodiagnostics

85 vues. Source : Azouz, N. P., et al., Investigation des granules sécrétoires des mastocytes ; de la biosynthèse à l’exocytose. J. Vis. Exp. (2015) Cette vidéo démontre l’exocytose des mastocytes induite par un réactif activateur qui comprend un ionophore calcique, initiant la libération de granules sécrétoires contenant la protéine rapporteure fluorescente NPY-mRFP. Par la suite, nous évaluons quantitativement l’exocytose en mesurant le NPY-mRFP libéré à l’aide d’un lecteur d...

Vidéo: Mesure de l’exocytose des mastocytes via la libération de la protéine fluorescente rouge monomère du neuropeptide Y - Concept

Imaging FITC-dextran as a Reporter for Regulated Exocytosis

Regulated exocytosis is a process by which cargo, which is stored in secretory granules (SGs), is released in response to a secretory trigger. Regulated exocytosis is fundamental for intercellular communication and is a key mechanism for the secretion of neurotransmitters, hormones, inflammatory mediators, and other compounds, by a variety of cells. At least three distinct mechanisms are known for regulated exocytosis: full exocytosis, where a single SG fully fuses with the plasma membrane, kiss-and-run exocytosis, where a single SG transiently fuses with the plasma membrane, and compound exocytosis, where several SGs fuse with each other, prior to or after SG fusion with the plasma membrane. The type of regulated exocytosis undertaken by a cell is often dictated by the type of secretory trigger. However, in many cells, a single secretory trigger can activate multiple modes of regulated exocytosis simultaneously. Despite their abundance and importance across cell types and species, the mechanisms that determine the different modes of secretion are largely unresolved. One of the main challenges in investigating the different modes of regulated exocytosis, is the difficulty in distinguishing between them as well as exploring them separately. Here we describe the use of fluorescein isothiocyanate (FITC)-dextran as an exocytosis reporter, and live cell imaging, to differentiate between the different pathways of regulated exocytosis, focusing on compound exocytosis, based on the robustness and duration of the exocytic events.

Here we detail a method for live cell imaging of regulated exocytosis. This method utilizes FITC-dextran, which accumulates in lysosome-related organelles, as a reporter. This simple method also allows distinguishing between different modes of regulated exocytosis in cells that are difficult to manipulate genetically.

L’imagerie FITC-dextran comme Reporter pour l’exocytose régulée

Regulated exocytosis is a process by which cargo, which is stored in secretory granules (SGs), is released in response to a secretory trigger. Regulated ...

Recherche

JoVE Journal

Biologie

Quantifying Spatiotemporal Parameters of Cellular Exocytosis in Micropatterned Cells

Live imaging of the pHluorin tagged Soluble N-ethylmaleimide-sensitive-factor Attachment protein REceptor (v-SNARE) Vesicle-associated membrane protein 7 (VAMP7) by total internal reflection fluorescence microscopy (TIRFM) is a straightforward way to explore secretion from the lysosomal compartment. Taking advantage of cell culture on micropatterned surfaces to normalize cell shape, a variety of statistical tools were employed to perform a spatial analysis of secretory patterns. Using Ripley&#8217;s K function and a statistical test based on the nearest neighbor distance (NND), we confirmed that secretion from lysosomes is not a random process but shows significant clustering. Of note, our analysis revealed that exocytosis events are also clustered in nonadhesion areas, indicating that adhesion molecules are not the only structures that can induce secretory hot spots at the plasma membrane. Still, we found that cell adhesion enhances clustering. In addition to precisely defined adhesive and nonadhesive areas, the circular geometry of these micropatterns allows the use of polar coordinates, simplifying analyses. We used Kernel Density Estimation (KDE) and the cumulative distribution function on polar coordinates of exocytosis events to identify enriched areas of exocytosis. In ring-shaped micropattern cells, clustering occurred at the border between the adhesive and nonadhesive areas. Our analysis illustrates how statistical tools can be employed to investigate spatial distributions of diverse biological processes.

Live imaging of lysosomal exocytosis on micropatterned cells allows a spatial quantification of this process. Morphology normalization using micropatterns is an outstanding tool to uncover general rules about the spatial distribution of cellular processes.

Quantification des paramètres spatiotemporal de l’exocytose cellulaire dans les cellules micropatternées

Live imaging of the pHluorin tagged Soluble N-ethylmaleimide-sensitive-factor Attachment protein REceptor (v-SNARE) Vesicle-associated membrane protein 7 ...

Automated Detection and Analysis of Exocytosis

Timelapse TIRF microscopy of pH-sensitive GFP (pHluorin) attached to vesicle SNARE proteins is an effective method to visualize single vesicle exocytic events in cell culture. To perform an unbiased, efficient identification and analysis of such events, a computer-vision based approach was developed and implemented in MATLAB. The analysis pipeline consists of a cell segmentation and exocytic-event identification algorithm. The computer-vision approach includes tools for investigating multiple parameters of single events, including the half-life of fluorescence decay and peak &#916;F/F, as well as whole-cell analysis of the frequency of exocytosis. These and other parameters of fusion are used in a classification approach to distinguish distinct fusion modes. Here a newly built GUI performs the analysis pipeline from start to finish. Further adaptation of Ripley&#39;s K function in R Studio is used to distinguish between clustered, dispersed, or random occurrence of fusion events in both space and time.

We developed automated computer vision software to detect exocytic events marked by pH-sensitive fluorescent probes. Here, we demonstrate the use of a graphical user interface and RStudio to detect fusion events, analyze and display spatiotemporal parameters of fusion, and classify events into distinct fusion modes.

Détection et analyse automatisées de l’exocytose

Timelapse TIRF microscopy of pH-sensitive GFP (pHluorin) attached to vesicle SNARE proteins is an effective method to visualize single vesicle exocytic ...

Measuring Mast Cell Exocytosis via Neuropeptide Y Monomeric Red Fluorescent Protein Release

Transcription

Measuring Mast Cell Exocytosis via Neuropeptide Y Monomeric Red Fluorescent Protein Release