We thank Dr. U. Ashery for the generous gift of cDNA. We thank Drs. G. Mass, L. Mittleman, M. Shaharbani, and Y.Zilberstein from the &#160;Sackler&#160;Cellular &#38; Molecular Imaging Center for their invaluable assistance with microscopy.&#160;This work was supported by the United States-Israel Binational Science Foundation (grant 2013263 to R. Sagi-Eisenberg, I. Hammel, and S.J.Galli) and grant 933/15 from the Israel Science Foundation, founded by the Israel Academy for Sciences (to R.Sagi-Eisenberg) and NIH grants U19 AI 104209 and R01 AR067145 (to S.J. Galli).

1. Preparations
<ol>
	<li>Preparation of RBL 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 ...

Here we describe how tracing the fluorescence of FITC-dextran loaded into SGs can be used to specifically capture compound exocytosis events. This was achieved by setting the microscope to acquire an image every 15 seconds, thus ensuring that only long-lasting events will be recorded over time, and therefore excluding short events that would correspond to full exocytosis or kiss-and-run exocytosis. To establish the method, we showed that knockdown of the Rab5 isoforms that are expressed in RBL cells, and are pivotal for ...

Regulated exocytosis is the primary mechanism by which readily made cargo is released from a secretory cell in response to a specific trigger. The cargo is pre-formed and sequestered into secretory vesicles, in which it is stored until a trigger relays the signal for the release of the SGs&#39; content. Different types of signals may result in different modes of regulated exocytosis, or different modes of regulated exocytosis may occur simultaneously. Three main modes of regulated exocytosis are known: full exocytosis, which involves full fusion of a single secretory granule with the plasma membrane; kiss-and-run exocytosis, which involves transient fusion of the secretory granule with the plasma membrane followed by its recycling; and compound exocytosis, which is characterized by homotypic fusion of several SGs prior (i.e., multigranular exocytosis) or sequential (i.e., sequential exocytosis) to fusion with the plasma membrane1. Compound exocytosis is considered the most extensive mode of cargo release2, as it allows for the fast secretion of cargo, including that from SGs that are located distal from the plasma membrane. Compound exocytosis has been documented in both exocrine and endocrine cells3,4,5,6,7,8,9, as well as in immune cells. In immune cells, such as eosinophils10,11,12 and neutrophils13, compound exocytosis allows the fast and robust release of mediators that are required to kill invading pathogens such as bacteria or parasites. Mast cells (MCs) deploy compound exocytosis for the efficient release of pre-stored inflammatory mediators during innate immune responses, anaphylaxis,&#160;and other allergic reactions14,15,16,17. Since the different modes of exocytosis may occur simultaneously18,19, it has become a challenge to distinguish between them in real time or to identify their respective fusion machineries, hence elucidating their underlying mechanisms.
Here we present a method, based on live cell imaging of cell loaded FITC-dextran, that allows real time tracking of exocytic events and distinguishing between their different modes. In particular, our method allows exclusive monitoring of compound exocytosis.
FITC-dextran is a conjugate of the pH-sensitive fluorophore FITC with the glucan polysaccharide dextran. Fluorescently labeled dextrans have been shown to enter the cell by micropinocytosis20,21 and macropinocytosis22,23. As endocytic compartments mature into lysosomes, it has been shown that FITC-dextran accumulates in the lysosome with no apparent degradation. However, since FITC is a highly pH-sensitive fluorophore24, and the lysosome lumen is acidic, FITC-dextran fluorescence quenches upon reaching the lysosome24. Thus, establishing dextrans as lysosome targeted cargo, taken together with the pH sensitivity of FITC, have laid the foundation for the use of FITC-dextran in studies of lysosome exocytosis25,26,27,28,29.
In several cell types, including MCs, neutrophils, eosinophils, cytotoxic T cells, melanosomes, and others, the SGs display lysosomal features and are classified as lysosome-related organelles (LROs) or secretory lysosomes30,31. Since LROs have an acidic luminal pH, FITC-dextran can be used to visualize their exocytosis, as a result of higher pH associated with the exteriorization of the LROs. Indeed, FITC-dextran has been used to monitor exocytosis in MCs18,32,33. In this method, FITC-dextran is added to the cell culture, taken up by the cells by pinocytosis and sorted into the SGs. As it is in lysosomes, FITC fluorescence is quenched in the SGs when they are within the cell. However, upon SG fusion with the plasma membrane and consequent exposure to the external milieu, the FITC-dextran regains its fluorescence as the SG pH rises, allowing the simple tracking of exocytic events by live cell microscopy. Here, we adjusted this method to enable unique tracking of compound exocytosis.
Two other methods have been used previously to track compound exocytosis. Electron microscopy was the first method to characterize exocytic structures that suggested the occurrence of different modes of exocytosis. In particular, observations of &#34;secretory tunnels&#34; in pancreatic acinar cells34 and MCs35,36,37 gave rise to the hypothesis of compound exocytosis. However, while the high resolution of electron microscopy has the power to reveal fused vesicles, it cannot track the dynamics of their fusion and hence can&#39;t define whether they correspond to SG fusion during compound exocytosis or fusion of recaptured granules following their endocytosis. This obstacle is overcome in other methods that can measure exocytosis in living cells, such as patch clamp measurements of the plasma membrane capacitance11,13,38,39 or amperometry40 of the media. However, patch clamping requires a special set-up and may not be suitable for all cell types. Amperometry measurements are able to track exocytosis only if the cargo is released in very close proximity to the electrode. Therefore, using live cell imaging offers an advantage over these methods, as it not only allows for real time tracking of exocytosis, but it also allows quick and simple acquisition of data from the whole cell.
The tracking of FITC-dextran by live cell microscopy also offers some advantages to other live cell imaging-based methods. For example, a widely used method is total internal reflection fluorescence microscopy (TIRFM) of cells loaded with a fluorescent SG probe or expressing a fluorescent protein-tagged SG cargo or membrane protein26,41,42,43. The strength of this method lies in its ability to monitor exclusively events that occur close to the plasma membrane (herein referred to as footprint), hence exocytic events. However, this is also the drawback of this method because only the cell fraction that is adjacent to the coverglass and close to the microscope lens can be imaged44. Whether such footprints indeed represent the entire cell membrane surface is still debatable45,46,47. In this regard, using a pH-sensitive dye such as FITC-dextran and a standard fluorescence microscope or a confocal microscope with an open pinhole allows imaging of the whole cell, thus capturing the total exocytic events that occur in that cell.
Additional pH-sensitive reporters that are used to study regulated exocytosis by whole cell imaging or TIRFM include SG cargo or SG membrane protein fused to phlourin, a pH-sensitive GFP variant. Examples include NPY-phlourin-, &#946;-hexoseaminidase-phlourin, and synapto-phluorin48,49,50,51,52. While expression of these probes may represent more closely the endogenous composition of the SGs, it entails transfection of the cells, and may therefore be less suitable for cells that are difficult to transfect. Therefore, when studying cells that are difficult to transfect or under experimental conditions that require multiple genomic manipulations, the use of a compound that can be simply supplemented into the cell culture medium, such as FITC-dextran, is advantageous. FITC-dextran also offers an advantage over acridine orange (AO), another pH-sensitive dye that has been used for the tracking of exocytosis by live cell microscopy53,54,55,56,57,58. AO has been shown to induce photolysis of vesicles that result in false flashes, which do not correspond to actual secretion processes27. In contrast, FITC-dextran reflects better secretion events, probably due to its low photo-induced production of reactive oxygen27.
Notably, an alternative approach for studying exocytosis is by tracking the influx of a dye, from the external medium into the SG through the fusion pore that opens during this process. In this case, the dye is added to the external medium alongside the secretory trigger. Then, when the fusion pore opens, the dye diffuses into the SG59,60. A clear advantage of this method is that it also offers the ability to estimate the fusion pore size, by the use of dyes of variable size. For example, dextrans of different molecular weight (MW), conjugated to different fluorophores, can be used as extracellular dyes whereby the maximum size of dextran that can penetrate the SG would correspond to the size of the fusion pore59,61,62,63,64. In addition, this approach does not require the use of a pH-sensitive probe. However, a significant disadvantage is that the signal to noise ratio is very low, since a large amount of dye is present in the media during acquisition of images, resulting in high background.
Overall, the use of FITC-dextran as a marker for exocytosis overcomes several drawbacks in previously reported methods, such as signal to noise ratio, toxicity, dynamic tracking and complexity.
Here we describe the use of FITC-dextran to monitor compound exocytosis in the RBL-2H3 mast cell line (herein referred to as RBL, primarily established by Eccleston et al.65 and further cloned by Barsumian et al.66), in response to immunoglobulin E (IgE)/antigen (Ag) activation.

<table>
	<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&#160;</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&#160;</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&#160;</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&#160;</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&#160;</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 
			airstream incubator</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 
			airstream incubator</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>

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.

Figure 1a represents schematically how FITC-dextran may act as a reporter for regulated exocytosis and recapitulate the different modes of exocytic events. First, cells are incubated with FITC-dextran, which is internalized by pinocytosis and sorted to the SGs. Since the SGs of MCs are LROs, their low pH dampens the fluorescence of FITC, shown here as black granules (A-C, I). When cells are triggered by a secretagogue and the SGs fuse with the plasma membrane...

Watch this Scientific Journal Video about Imaging FITC-dextran as a Reporter for Regulated Exocytosis at JoVE.com

Imaging FITC-dextran as a Reporter for Regulated Exocytosis

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.

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

This method can help answer key questions in the research of regulated exocytosis, by allowing researchers to distinguish between the different modes of exocytosis in the living cell. The main advantage of this technique is that it is simple and requires minimal perturbation to the cells. Though this method can provide insight into mast cell exocytosis, it can also be applied to other secretory cells such as neutrophils and eosinophils.To begin, prepare a stock solution of 20x Tyrode's buffer according to the text protocol. Next, mix three milligrams of FITC-dextran powder with three milliliters of culture media. Filter the dissolved FITC-dextan with a cellulose acetate syringe filter unit.Then, add mouse IgE to a concentration of one microgram per milliliter. One day prior to imaging, aspirate media from a culture dish and replace it with two milliliters of Trypsin EDTA Solution B.Then, incubate the dish for five to 10 minutes. Once the cells have detached, add two milliliters of culture media to the dish to neutralize the Trypsin.Then, use a hemocytometer to count the RBL cells. Adjust the volume of the suspension with culture media to achieve a concentration of 750, 000 cells per milliliter. Next, 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'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'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's buffer. After repeating the washing process two times, replenish the chamber with 300 microliters of Tyrode's buffer. Next, place the chambered coverglass in the incubator chamber of a microscope and insure 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 bidirectional, 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. In this protocol, FITC-dextran was used as a reporter for live cell imaging of regulated exocytosis.Imaging FITC-dextran release allows capturing of the sequential nature of compound exocytosis. After the addition of ammonium chloride to the media, FITC-dextran becomes visible and is co-localized with the mRFP-tagged neuropeptide Y, as secretory granular reporter in RBL cells. While attempting this procedure, it is important to minimize toxicity to the cells while filming by minimizing the exposure of the cells to the laser.This can be achieved through using the low laser power and short acquisition time. Since compound exocytotic events are longer lived in comparison to other regulated exocytic events, we can categorically capture compound exocytosis by setting a long time interval between acquisitions.

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12.4K vistas.  Tel Aviv University. This method can help answer key questions in the research of regulated exocytosis, by allowing researchers to distinguish between the different modes of exocytosis in the living cell. The main advantage of this technique is that it is simple and requires minimal perturbation to the cells. Though this method can provide insight into mast cell exocytosis, it can also be applied to other secretory cells such as neutrophils and eosinophils.To begin, prepare a stock solution of 20x Tyrode's...