an in vitro technique to visualize exocytosis of secretory granules in mast cells

An In Vitro Technique to Visualize Exocytosis of Secretory Granules in Mast Cells

Add 10 microliters of the cell suspension to a chambered coverglass with fresh FITC-dextran supplemented culture media, and grow the RBL cells overnight. First, prepare a final Tyrode&#39;s buffer solution according to the text protocol. Then, prepare a 20x secretagogue reagent in the freshly-made buffer.
Dissolve ammonium chloride powder in Tyrode&#39;s buffer to create a 400-millimolar ammonium chloride solution. Next, aspirate the media from the chambered coverglass, and replace it with 300 microliters of pre-warmed Tyrode&#39;s buffer. After repeating the washing process two times, replenish the chamber with 300 microliters of Tyrode&#39;s buffer.
Next, place the chambered coverglass in the incubator chamber of a microscope, and ensure the chamber is stable. Turn on the fluorescent light source and select the appropriate fluorescence filter. Once the region of interest is in focus in the middle of the field of view, turn off the light source.
Then, turn on the 488-nanometer laser with the emission gathered around 500 to 550 nanometers. Next, calibrate the time interval between image acquisitions. Set the scan direction to &#34;Bi-directional.&#34; Do not allow for averaging. Open the pinhole, and set the resolution at 512 by 512.
Next, image the cells for the appropriate duration for the specific cell type or secretagogue. Then, add 16 microliters from the secretagogue solution to the chamber and continue filming. Finally, to confirm the presence and localization of FITC-dextran to the secretory granules, gently add 16 microliters of ammonium chloride solution to the chamber.

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JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Imaging and Visualization Techniques

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1. Preparations
<ol>
	<li>Preparation of RBL (RBL-2H3 mast cell line) culture media
	<ol>
		<li>Mix 500 mL of low glucose Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) with 56 mL of fetal bovine serum (FBS), 5.5 mL of penicillin-streptomycin-nystatin solution, 5.5 mL of L-Glutamine 200 mM solution. This results in low glucose DMEM supplemented with 10% FBS, 100 &#181;g/mL streptomycin, 100 units/mL penicillin, 12 units/mL nystatin, and 2 mM L-glutamine.</li>
		<li>Filter the media by using a top-vacuum filter of 0.22 &#181;m pore size and store at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Maintenance of RBL cells.
	<ol>
		<li>Grow RBL cells to a maximum confluency of 90% in a 10 cm dish. If the cell culture is healthy, the cells should have a spindle shape with occasional protrusions.</li>
		<li>For cell splitting, detach the cells from the dish by aspirating the media and replacing it with 2 mL of trypsin/EDTA solution B. Incubate for 5-10 minutes in a humidified atmosphere of 5% CO2 at 37 &#176;C.</li>
		<li>Once the cells have detached, neutralize the trypsin by adding 2 mL of culture media, using a pipette, and split the cells in a 1:2 - 1:10 ratio.</li>
	</ol>
	</li>
	<li>Preparation of 20x Tyrode&#39;s buffer
	<ol>
		<li>Prepare a stock 20x solution of 54 mM KCl, 20 mM MgCl2, 2.74 M NaCl, and 8 mM NaH2PO4 in double distilled water (DDW). Mix well and store at 4 &#176;C.</li>
	</ol>
	</li>
</ol>
2. Culture of RBL Cells for Live Cell Microscopy
<ol>
	<li>Preparation of FITC-dextran solution.
	<ol>
		<li>Mix 1 mg of fluorescein isothiocyanate (FITC)-dextran powder (150 K) per 1 mL of culture media (see step 1.1.1). For a full chambered coverglass, prepare 3 mL.</li>
		<li>Using a cellulose acetate syringe filter unit with 0.22 &#181;m pore size, filter the dissolved FITC-dextran.</li>
		<li>Add mouse immunoglobulin E (IgE) to a concentration of 1 &#181;g/mL.</li>
	</ol>
	</li>
	<li>Seeding RBL cells for imaging
	<ol>
		<li>The day before imaging, aspirate the media from the culture dish and replace it with 2 mL of trypsin/ethylenediaminetetraacetic acid (EDTA) solution B. Incubate for 5 - 10 minutes in a humidified atmosphere of 5% CO2 at 37 &#176;C. Once the cells have detached, neutralize the trypsin by adding 2 mL of culture media.</li>
		<li>Count RBL cells using a hemocytometer and adjust the volume accordingly with culture media to get a cell concentration of 7.5 x 105/mL.</li>
		<li>Add 10 &#181;L of the cell suspension to a chambered coverglass pre-filled with fresh FITC-dextran supplemented culture media (this results in the seeding of 7.5 x 103 cells in a chamber).</li>
		<li>Grow RBL cells overnight in a humified atmosphere of 5% CO2 at 37 &#176;C. The cells should remain at a sub-confluent level in order to make sure that the cells are separated and that it is easy to identify each cell individually under the microscope.</li>
	</ol>
	</li>
	<li>Transfection of RBL cells - optional.
	<ol>
		<li>For imaging exocytic events in combination with other fluorescently tagged proteins, see transfection protocol for RBL cells in Azouz et al.</li>
	</ol>
	</li>
</ol>
3. Live Cell Microscopy of Exocytosis
<ol>
	<li>Preparation of solutions:
	<ol>
		<li>Prepare a final Tyrode&#39;s buffer solution by diluting the stock solution in DDW in a 1:20 dilution and supplement with 20 mM Hepes pH 7, 1.8 mM CaCl2, 1 mg/mL bovine serum albumin (BSA), and 5.6 mM glucose.</li>
		<li>Freshly prepare a 20x secretagogue reagent in Tyrode&#39;s buffer [1 &#181;g/mL dinitrophenyl conjugated to human serum albumin (DNP-HAS (Ag)) in our case, for a 50 ng/mL 1x concentration].</li>
		<li>Prepare a 400 mM ammonium chloride solution by dissolving the powder in Tyrode&#39;s buffer.</li>
	</ol>
	</li>
	<li>Preparation of the cells.
	<ol>
		<li>Wash the chambered coverglass 3 times by aspirating the media from the chamber and refilling it with 300 &#181;L of Tyrode&#39;s buffer, prewarmed to 37 &#176;C. Finally, replenish the chamber with 300 &#181;L of Tyrode&#39;s buffer, prewarmed to 37 &#176;C.</li>
		<li>Place the chambered coverglass in the microscope&#39;s incubator chamber. Make sure that the chamber is stable.</li>
	</ol>
	</li>
	<li>Setting up the microscope
	<ol>
		<li>To choose a region of interest to track, turn on the fluorescent light source (traditionally a mercury lamp) and choose the appropriate fluorescence filter (choose the filter for green fluorophores for viewing FITC-dextran). Once the region of interest is in focus and is in the middle of the field of view, turn off the light source in order to avoid photo-bleaching and toxicity. 
		&#8203;NOTE: Some of the FITC-dextran incorporated in the cells may retain fluorescence. This is because FITC-dextran may also be sorted into non-lysosomal compartments such as endosomes.</li>
		<li>Turn on the appropriate lighting for FITC excitation. If using a laser-based microscope, turn on the 488 nm laser. Emission should be gathered around 500 - 550 nm (FITC emission peaks at 510 - 520 nm).</li>
		<li>When using a confocal microscope, open the pinhole to the maximum. This will allow the use of lower laser power to avoid bleaching and toxicity and will ensure the capture of exocytosis events from all planes of the cell.</li>
		<li>Calibrate the time interval between image acquisitions.
		<ol>
			<li>Different cells may vary in their kinetics of regulated exocytosis. For specific imaging of compound exocytosis, we recommend a time interval of at least 5 seconds. However, as a rule of thumb, to allow for fast imaging, make sure that the acquisition time of a single frame is fast.</li>
			<li>When using a laser-scanning confocal microscope, set the scan direction to bi-directional, do not allow for averaging, and set the resolution at 512 x 512 (recommended; the latter two provisions will also help to minimize bleaching and toxicity).</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Imaging of exocytosis.
	<ol>
		<li>Image cells for desired durations, depending on the type of cell or secretagogue. In RBL cells triggered with IgE/Ag, most exocytic events occur within 15 - 20 min of activation.</li>
		<li>For activation of the cells, add 16 &#181;L from the 20x secretagogue solution to the chamber.</li>
	</ol>
	</li>
	<li>Confirming FITC-dextran presence in the cells.
	<ol>
		<li>To confirm the presence and localization of FITC-dextran to the SGs, add 16 &#181;L of ammonium chloride solution (be careful not to move the chamber in order not to lose focus) to the chamber gently (this will make a final concentration of 20 mM). This will induce alkalization of the SGs and dequenching of FITC fluorescence, which will occur within seconds.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>DMEM low glucose</td>
			<td>Biological Inductries</td>
			<td>01-050-1A</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco</td>
			<td>12657</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen-strep-nystatin solution</td>
			<td>Biological Inductries</td>
			<td>03-032-1B</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine 200 mM solution</td>
			<td>Biological Inductries</td>
			<td>03-020-1A</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC dextran 150K</td>
			<td>Sigma-Aldrich</td>
			<td>46946-500MG-F</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin/EDTA Solution B</td>
			<td>Biological Inductries</td>
			<td>03-052-1A</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr>
			<td>Top-vacuum filter of 0.22 &#181;m pore size</td>
			<td>Sigma-Aldrich</td>
			<td>CLS430769-1EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Cellulose acetate syringe filter unit, 0.22 &#181;m pore size</td>
			<td>Sartorius</td>
			<td>16534K</td>
			<td></td>
		</tr>
		<tr>
			<td>Chambered coverglass</td>
			<td>Thermo scientific</td>
			<td>155411</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>Bovine serum albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A4503</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-DNP monoclonal IgE</td>
			<td>Sigma-Aldrich</td>
			<td>D8406</td>
			<td></td>
		</tr>
		<tr>
			<td>DNP-HSA (Ag)</td>
			<td>Sigma-Aldrich</td>
			<td>A6661</td>
			<td>Avoid direct light exposure</td>
		</tr>
		<tr>
			<td>Hepes buffer 1M, pH 7.4</td>
			<td>Biological Inductries</td>
			<td>03-025-1B</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>MERK</td>
			<td>102382</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>BDH Laboratories</td>
			<td>284515V</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium chloride</td>
			<td>MERK</td>
			<td>1145</td>
			<td></td>
		</tr>
		<tr>
			<td>PIPES dipotassium salt</td>
			<td>Sigma-Aldrich</td>
			<td>108321-27-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium acetate hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>114460-21-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium acetate tetrahydrate</td>
			<td>Sigma-Aldrich</td>
			<td>M5661</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium glutamate (L-Glutamic acid potassium salt monohydrate)</td>
			<td>Sigma-Aldrich</td>
			<td>G1501</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>MERK</td>
			<td>6346</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>MERK</td>
			<td>106404</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>MERK</td>
			<td>105833</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>MERK</td>
			<td>104936</td>
			<td></td>
		</tr>
		<tr>
			<td>Electroporator</td>
			<td>BTX</td>
			<td>ECM830</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope, Zeiss</td>
			<td>Zeiss</td>
			<td>LSM 5 Pascal, Axiovert 200M</td>
			<td>Used in Figure 3. Equipped with an electronic temperature-controlled</td>
		</tr>
		<tr>
			<td>airstream incubator</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope, Zeiss</td>
			<td>Zeiss</td>
			<td>LSM 800, Axio Observer.Z1 /7</td>
			<td>Used in Figure 2. Equipped with a GaAsP detector an electronic temperature-controlled</td>
		</tr>
		<tr>
			<td>airstream incubator</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope, Leica</td>
			<td>Leica</td>
			<td>SP5</td>
			<td>Used in Figure 1. Equipped with a leica HyD detector and an top-stage incubator (okolab)</td>
		</tr>
		<tr>
			<td>RBL-2H3 cells</td>
			<td></td>
			<td></td>
			<td>RBL-2H3 cells were cloned in the lab of Reuben P. Siraganian. See reference 67</td>
		</tr>
	</tbody>
</table>

Source: Klein, O., et al. <a href="https://app.jove.com/v/57936">Imaging FITC-dextran as a Reporter for Regulated Exocytosis</a> J. Vis. Exp. (2018).
This video demonstrates a technique to visualize exocytosis in mast cells. The cells are incubated with FITC-dextran &#8212; a fluorescent reporter &#8212; which is taken up by cells inside secretory granules (SGs), where the acidic pH quenches its fluorescence. Upon stimulation of granule exocytosis, the medium&#39;s higher pH induces reporter fluorescence inside the fused SGs, which is visualized using a fluorescence microscope.

1. Preparations

	Preparation of RBL (RBL-2H3 mast cell line) culture media
	
		Mix 500 mL of low glucose Dulbecco&#39;s modified Eagle&#39;s medium ...

Take a chambered coverglass containing mast cells &#8212; immune cells with secretory granules, or SGs, containing inflammatory mediators.
Add IgE antibodies along with a pH-sensitive fluorescent reporter, and incubate.
The antibodies bind to Fc receptors on mast cells. The reporters get internalized via pinocytosis, and the resulting endosomes fuse with SGs.
Remove the media. Add an antigen containing multiple IgE-binding epitopes; place the slide under a fluorescence microscope, and apply the required excitation wavelength.
The acidic pH of SGs quenches reporter fluorescence.
The antigen binds to IgE-receptor complexes on the cell surface, crosslinking them.
Antigen-mediated receptor aggregation triggers SGs to migrate toward the plasma membrane.
SNARE proteins on the plasma and SG membrane mediate membrane fusion and the discharge of SG cargo, termed exocytosis.
The higher pH of the medium causes SG alkalization, inducing the reporters to regain fluorescence.
Incoming SGs fuse with the membrane-fused ones. Alkalization of the second SG mediates fluorescence recovery, confirming compound exocytosis.

32 visualizações. Uma técnica in vitro para visualizar a exocitose de grânulos secretores em mastócitos

Vídeo: Uma técnica in vitro para visualizar a exocitose de grânulos secretores em mastócitos - Experimento

Monitoring the Effect of Osmotic Stress on Secretory Vesicles and Exocytosis

Amperometry recording of cells subjected to osmotic shock show that secretory cells respond to this physical stress by reducing the exocytosis activity and the amount of neurotransmitter released from vesicles in single exocytosis events. It has been suggested that the reduction in neurotransmitters expelled is due to alterations in membrane biophysical properties when cells shrink in response to osmotic stress and with assumptions made that secretory vesicles in the cell cytoplasm are not affected by extracellular osmotic stress. Amperometry recording of exocytosis monitors what is released from cells the moment a vesicle fuses with the plasma membrane, but does not provide information on the vesicle content before the vesicle fusion is triggered. Therefore, by combining amperometry recording with other complementary analytical methods that are capable of characterizing the secretory vesicles before exocytosis at cells is triggered offers a broader overview for examining how secretory vesicles and the exocytosis process are affected by osmotic shock. We here describe how complementing amperometry recording with intracellular electrochemical cytometry and transmission electron microscopy (TEM) imaging can be used to characterize alterations in secretory vesicles size and neurotransmitter content at chromaffin cells in relation to exocytosis activity before and after exposure to osmotic stress. By linking the quantitative information gained from experiments using all three analytical methods, conclusions were previously made that secretory vesicles respond to extracellular osmotic stress by shrinking in size and reducing the vesicle quantal size to maintain a constant vesicle neurotransmitter concentration. Hence, this gives some clarification regarding why vesicles, in response to osmotic stress, reduce the amount neurotransmitters released during exocytosis release. The amperometric recordings here indicate this is a reversible process and that vesicle after osmotic shock are refilled with neurotransmitters when placed cells are reverted into an isotonic environment.

Osmotic stress affects exocytosis and the amount of neurotransmitter released during this process. We demonstrate how combining electrochemical methods together with transmission electron microscopy can be used to study the effect of extracellular osmotic pressure on exocytosis activity, vesicle quantal size, and the amount of neurotransmitter released during exocytosis.

O efeito do estresse osmótico em vesículas secretoras e exocitose de monitoramento

Amperometry recording of cells subjected to osmotic shock show that secretory cells respond to this physical stress by reducing the exocytosis activity ...

JoVE Journal

Neurociência

Confocal Imaging of Neuropeptide Y-pHluorin: A Technique to Visualize Insulin Granule Exocytosis in Intact Murine and Human Islets

Insulin secretion plays a central role in glucose homeostasis under normal physiological conditions as well as in disease. Current approaches to study insulin granule exocytosis either use electrophysiology or microscopy coupled to the expression of fluorescent reporters. However most of these techniques have been optimized for clonal cell lines or require dissociating pancreatic islets. In contrast, the method presented here allows for real time visualization of insulin granule exocytosis in intact pancreatic islets. In this protocol, we first describe the viral infection of isolated pancreatic islets with adenovirus that encodes a pH-sensitive green fluorescent protein (GFP), pHluorin, coupled to neuropeptide Y (NPY). Second, we describe the confocal imaging of islets five days after viral infection and how to monitor the insulin granule secretion. Briefly, the infected islets are placed on a coverslip on an imaging chamber and imaged under an upright laser-scanning confocal microscope while being continuously perfused with extracellular solution containing various stimuli. Confocal images spanning 50 &#181;m of the islet are acquired as time-lapse recordings using a fast-resonant scanner. The fusion of insulin granules with the plasma membrane can be followed over time. This procedure also allows for testing a battery of stimuli in a single experiment, is compatible with both mouse and human islets, and can be combined with various dyes for functional imaging (e.g., membrane potential or cytosolic calcium dyes).

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.

Confocal da imagem latente do Neuropeptide Y-pHluorin: uma técnica para visualizar a exocitose de grânulo de insulina em ilhotas murino e humanas intactas

Insulin secretion plays a central role in glucose homeostasis under normal physiological conditions as well as in disease. Current approaches to study ...

Biologia

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.

Detecção e Análise Automatizada de Exocistose

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

An In Vitro Technique to Visualize Exocytosis of Secretory Granules in Mast Cells

Transcrição

An In Vitro Technique to Visualize Exocytosis of Secretory Granules in Mast Cells