We thank Dr. U. Ashery for the gift of NPY-mRFP cDNA. We thank Drs. M. J. Kofron, L. Mittleman, M. Shaharbani, and Y. Zilberstein for invaluable assistance with microscopy and image analyses. We also thank Dr. Joseph Orly for critical reading of this manuscript. &#160;This work was supported by a grant from the Israel Science Foundation, founded by&#160;the Israel Academy for Sciences (1139/12 to R.S-E.).

1. Preparation of RBL Cell Culture Media
<ol>
	<li>Mix 500 ml of low glucose Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) with 56&#160;ml of fetal bovine serum (this makes 10% FBS), and then add 5.5 ml of penicillin streptomycin (this makes ~1% PEST).</li>
	<li>Filter the media by using 500-1,000 ml Bottle-Top Vacuum Filters with 0.22 &#956;m pore size and store at 4 &#176;C.</li>
</ol>
2. Culture of RBL Cells
<ol>
	<li>Grow RBL cells in a humidified atmosphere of 5% CO2 at 37 &#176;C in either plates or flasks. RBL cells that are growing in 10 cm plates should be supplemented with 10 ml medium and form confluent monolayer of ~107 cells/plate.</li>
	<li>When the cell layer is approaching confluence, replate&#160;the culture at a lower density.
	<ol>
		<li>Remove the culture medium from the plate/flask using a sterile pasteur pipette connected to vacuum.</li>
		<li>Rinse the cell monolayer with prewarmed (37 &#176;C) 0.25% trypsin/EDTA that covers the whole monolayer (the volume depends on the size of culture plate/flask; 2 ml for 10 cm plate). This will detach the cells.</li>
		<li>Alternatively, rinse the cell monolayer with Phosphate buffered saline (PBS), remove the PBS and add prewarmed (37 &#176;C) trypsin/EDTA. This step detaches the cells faster.</li>
		<li>Place the plate/flask in an incubator at 37 &#176;C (no more than 10 min).</li>
		<li>Inspect the cells under the optical microscope to check if they have detached. If the cells did not detach after 10 min, gently tap on the plate/flask.</li>
		<li>Add medium to the culture plate/flask to suspend the cells (the volume of the medium should be at least 2-fold larger than the volume of the trypsin solution).</li>
		<li>Pellet the cells by centrifugation for 3 min at 200 g at 25 &#176;C.</li>
		<li>Resuspend the cells in medium. Dilute the cells by 1:10 and seed in a new culture dish. Incubate the cells for 48-72 hr, until they reach approximately 90% confluence.</li>
		<li>Repeat the same procedure (section 2.2.1-2.2.8) for up to 3 months (30 passages).</li>
	</ol>
	</li>
</ol>
3. Preparation of Transfection Media.
<ol>
	<li>Prepare a 100 mM solution of K-Pipes pH 7, 1 mM solution of Ca2+ acetate and 100 mM solution of Mg2+ acetate.</li>
	<li>Dilute the 100 mM solution of K-Pipes pH 7 into 20 mM using DMEM media, add the 1 mM solution of Ca2+ acetate to a final concentration of 10 &#956;M and 100 mM solution of Mg2+ acetate to a final concentration of 2 mM.</li>
	<li>Weigh&#160;Potassium glutamate and add to the solution to a final concentration of 128 mM, mix well and keep at 4 &#176;C until use. Keep the remaining solutions (i.e., K-Pipes, Ca2+ acetate, Mg2+ acetate) at -20 &#176;C.</li>
</ol>
4. Transfection of RBL Cells
<ol>
	<li>Remove the culture medium from the plate/flask using a sterile pasteur pipette connected to vacuum.</li>
	<li>Rinse the cell monolayer with prewarmed (37 &#176;C) trypsin/EDTA that should cover the whole monolayer. Place the plate/flask in an incubator at 37 &#176;C (no more than 10 min). Check if the cells have detached using the optical microscope.</li>
	<li>Add medium to the culture plate/flask to suspend the cells (the volume of the media should be at least 2-larger more than the volume of the trypsin solution).</li>
	<li>Count the cell number using hemocytometer.</li>
	<li>Pellet 1.5 &#215; 107 cells by centrifugation for 3 min at 200 x g at 25 &#176;C. Discard the supernatants and add 280 &#181;l of transfection medium. Transfer the reaction mix into 4 mm cuvette.</li>
	<li>Add 20 &#956;g of NPY-mRFP plasmid and either 30 &#956;g of control empty plasmid or a tested plasmid. The final volume of the reaction mix should be 300 &#956;l.</li>
	<li>Immediately place on ice for 10 min. Wipe the cuvette from residual water and proceed to electroporation at 300 V for 9 msec.</li>
	<li>For measurements of exocytosis, replate the cells immediately in 24 well tissue culture dishes containing 300 &#956;l medium. Add 8 &#956;l of the reaction mix (4 &#215; 105 cells) to each well. Add 4 &#215; 105 non transfected cells to additional wells for control. Prepare enough wells for to have at least duplicate wells for each treatment for every transfection.</li>
	<li>For time lapse microscopy, replate the cells immediately in an 8-well chamber borosilicate coverglass system containing 80 &#956;l medium. Add 1.5 &#956;l of the reaction mix (7.5 &#215; 104 cells) to each chamber. (Try to avoid using the chambers at the edge of the coverglass, the chambers at the center of the coverglass are easier to image). 
	NOTE: Importantly, not all the cells response to a trigger and occasionally the time-lapse microscopy is abrogated due to loss of focus and drift&#160;of the 8-well chamber borosilicate coverglass system. Therefore, prepare at least 8 chambers for each treatment for each transfection.</li>
	<li>For sensitizing the cells for Fc&#949;RI mediated activation, add 1 &#181;g/ml of mouse DNP specific monoclonal IgE to the media (incubate the cells with IgE for at least 2 hr).</li>
	<li>After 18-24 hr use a fluorescent microscope to confirm that the cells express the plasmids. The excitation and emission wavelengths of mRFP fluorescence are: &#955;Ex 584, &#955;Em 607 nm. NPY-mRFP should appear in vesicular structures which correspond to the SGs.&#160;If the second gene of interest is fused to a fluorescent tag use a fluorescent microscope to confirm the co-expression of both plasmids at the same cells. For example, if the tested gene is fused to GFP, The excitation and emission wavelengths of GFP fluorescence are: &#955;Ex 488, &#955;Em 507 nm. The GFP tagged protein should appear at the same cells that express NPY-mRFP.</li>
	<li>For exocytosis measurements proceed to step 5 and for time lapse microscopy, proceed to step 6.</li>
</ol>
5. 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 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 dilution of Tyrode 20x in DDW and mix well. This step is for preparation of 1x Tyrode buffer.</li>
		<li>Aliquot the 1x Tyrode buffer and store at -20 &#176;C. Avoid repeated freezing and thawing.</li>
	</ol>
	</li>
	<li>Remove the culture medium from the 24 well plate 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+&#160;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.&#160;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 from 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 preparation of cell lysates that contain the remaining of NPY-mRFP that was not released from the cells). Collect the cell lysates and transfer to a 96 well plate, place on ice and avoid from 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 measure arbitrary fluorescence units (AFU). AFU values depend on the machine and its sensitivity and the transfection efficiency.
	<ol>
		<li>Set the autofluorescence of nontransfected RBL cells as blank. Divide the AFU of each supernatant to the total fluorescence (AFU of the supernatants + AFU of the corresponding lysate) and multiply by 100.</li>
	</ol>
	</li>
</ol>
6. Time-lapse Microscopy of Exocytosis
<ol>
	<li>Seed 7.5 &#215; 104 of the transfected cells/chamber in an 8-well chamber borosilicate coverglass system. After 18-24 hr, remove the culture medium from the chambers and wash 3 times with Tyrode buffer</li>
	<li>Add 72 &#956;l of Tyrode buffer to each chamber. Dilute the activating reagents in Tyrode buffer to 10x concentration.</li>
	<li>Use a confocal fluorescence microscope equipped with a heated chamber (37 &#176;C) and CO2 controller (4.8%) and a 40X or 63X objective. Turn on the microscope systems: mercury lamp, computer, and lasers. Make sure that the heated chamber is at the right temperature before starting the experiment.</li>
	<li>Place the chamber in the heated chamber and make sure that the chamber is installed correctly and stable.</li>
	<li>Turn on the fluorescence light according to the relevant fluorophore and visualize transfected cells. Turn off fluorescence once a cell of interest is in the field in order to minimize bleaching and cytotoxicity. It is important that this field will contain about 2-3 transfected cells. The transfected cells should be well spread but not touching each other.</li>
	<li>Adjust the laser power (depending on the microscope) to minimize noise and oversaturation as well as toxicity, and set the gain and offset to modify the signal to noise ratio. Scan fast in order to minimize the duration of laser exposition (average 2).</li>
	<li>Adjust the pinhole size to a maximum, this enables decreasing the laser power and to maintain images focused for a long period of time. If desired, set the parameters for the Z stack to reconstruct the image in three dimensional, adjust the pinhole size to 1 airy unit (AU) that gives the best signal to noise ratio and acquire successive scanning of two-dimensional confocal optical slices in the z-axes with optical slices &#8804;0.7 &#956;m.</li>
	<li>Set the interval time between each acquisition to 15-30 sec and the duration of total acquisition to 15&#160;min and start acquiring images. After 5&#160;min of acquisition pause the time series. Add 8 &#956;l of the 10x trigger and continue the acquisition immediately.</li>
	<li>Save the pictures, perform deconvolution using a deconvolution software and reconstruct the stacks to three dimensional images and to a movie using Imaris software.</li>
</ol>
7. Image Analyses
<ol>
	<li>Import the data from the deconvoluted time series images that were obtained by the confocal fluorescence microscope to Imaris software. This software reads more than 40 microscopy files. If the software cannot read the files; convert the files to tif files and import.</li>
	<li>To add time points to the end of an open data set press edit -&#62; Add Time Points and import the new data set.</li>
	<li>Create surface of the mRFP channel using surface wizard option. Choose the default algorithm. Choose the channel of NPY-mRFP. Mark the smoothing option to reduce noise. To avoid loss of small details reduce the area detail level to 0.05 &#956;m.</li>
	<li>Set the intensity threshold. New gray surface will be displayed. Go to &#8220;Settings&#8221; and switch the style from &#8220;Center point&#8221; to &#8220;Surface&#8221;.</li>
	<li>Go to the statistic tab in the properties of selected object, select the detailed tab and the specific value such as fluorescence intensity, volume etc.</li>
	<li>Review the data and confirm that the values are compatible with the images. For example, two or more adjacent granules might be measured as one bigger granule. For quantifying the average granule size divide the size of the merged granules to the actual number of granules.</li>
	<li>Export the desired data sets to Excel files and analyze the data.</li>
</ol>f

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The authors have no financial conflicts of interest.

We describe an innovative strategy that combines quantification of MCs exocytosis and four (x, y, z, t) dimension quantifications by time-lapsed three-dimensional imaging of the SGs in living cells using a reporter gene for exocytosis. This technique enables screening of families of proteins for their impact on MC function such as monitoring SGs starting as early as their exit from the Golgi through their maturation, acquisition of exocytosis competence and degranulation. The combination of&#160;measurements of exocytosi...

Mast Cells (MC) are immune cells that are best known for their involvement in allergic and inflammatory reactions such as arthritis, asthma, eosinophilic esophagitis, chronic dermatitis and anaphylactic shock 1,2 as well as other pathologies including coronary artery disease 3,5 and cancer 3,4. In addition, MCs play important roles in innate and adaptive immunity, both in host defense against bacteria and parasites and by suppression of immune responses, for example inducing allograft tolerance 5,6 .
MCs originate from the bone marrow, developing from CD34+/CD117+ pluripotent progenitor cells 7. Committed bone marrow MC&#160;progenitors are released into the bloodstream and migrate into the peripheral tissues localizing predominantly within connective tissues and epithelial surfaces 8. Maturation and terminal differentiation are eventually achieved under the influence of cytokines within the surrounding milieu 8,9 .
MCs can be activated by an allergen (antigen, Ag), whose encounter resulted in the generation of immunoglobulin E (IgE) type antibodies. Binding of such IgE to the MC&#8217;s Fc&#949;RI receptors, followed by cross-linking of cell bound IgE upon re-exposure to the same Ag, results in Fc&#949;RI aggregation and initiation of a signaling cascade that culminates in cell degranulation [reviewed in 10,11]. MCs are also activated, independently of IgE, by neuropeptides 5,12, toxins 13 , bacterial and viral antigens 14,15, a number of positively charged peptides collectively referred to as basic secretagogues, immune cells and cytokines 5,13,12,16,17. MCs are also activated by many of their own released mediators, which further amplify the inflammatory response.
MCs are packed with secretory granules (SGs) that contain immunoregulatory mediators, including vasoactive amines, such as histamine and serotonin (in rodents), proteoglycans, proteases, such as chymase and tryptase, vascular endothelial growth factor and several cytokines and chemokines 8,9. These mediators are &#8220;ready to go&#8221; and once MCs are activated by an appropriate stimulus, these mediators are released from the cells by regulated exocytosis (degranulation) in a matter of seconds to minutes 18,19. This initial event is followed by the de novo synthesis and release of a large array of biologically potent substances, including arachidonic acid metabolites, multiple cytokines and chemokines 20,21,22. Release of newly synthesized products occurs independently of SG release. Collectively, these mediators initiate early and late phase inflammatory and allergic responses. Therefore, understanding the mechanisms accounting for MC activation and degranulation are both of theoretical and clinical importance.
The difficulty to genetically manipulate primary and cultured MCs has hampered the attempts to elucidate the mechanisms underlying MC degranulation, which remained poorly resolved. To overcome this problem we developed a reporter based assay by co-transfecting the mucosal mast cell line, rat basophilic leukemia (RBL)-2H3 (herein referred to as RBL) or bone marrow derived MCs&#160;(BMMCs) 30 with a gene of interest and Neuropeptide Y (NPY) fused to monomeric RFP (mRFP), as a SG reporter.
NPY was previously shown to recapitulate the behavior of endogenous SG markers in other systems. Moreover, because mRFP fluorescence is pH insensitive, expression of NPY-mRFP allows visualization of the acidic SGs as well as quantitative assessment of exocytosis by using 96-well plates and a fluorescence plate reader. We have shown that NPY-mRFP is delivered to the acidic SGs of RBL cells and BMMCs and is released from the cells in a regulated fashion alongside the endogenous SG&#160;cargo (i.e., &#946;-hexosaminidase and serotonin) 30,32 . This protocol provides a high-resolution imaging-based methodology that allows screening genes of interest for their phenotypic and functional impact on SG characteristics and degranulation in RBL cells 32. Specifically, this protocol allows real time tracking of MC SGs and quantification of their area or volume size, their number, kinetics of assembly, their movement along the cell cytoskeleton and their ultimate fusion with the plasma membrane under different conditions. For example, sensitizing the cells with DNP-specific IgE and triggering the cells with a multivalent Ag (DNP conjugated serum albumin) under different perturbations (i.e., knockdown of genes of interest, over expression of wt or mutant genes, or pharmacological manipulations) and comparing to control cells.

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	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Name of Material/ Equipment</td>
			<td style="width:128px;">Company</td>
			<td style="width:139px;">Catalog Number</td>
			<td style="width:156px;">Comments/Description</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:421px;">DMEM</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td>D6046-500ML</td>
			<td style="width:156px;">Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Fetal Bovine Serum</td>
			<td style="width:128px;">GE health care Life sciences</td>
			<td>SH30071.01</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin-Streptomycin</td>
			<td style="width:128px;">Life technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Cellulose acetate membrane, pore size 0.22&#160;&#956;m</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td height="20" style="height:20px;width:139px;">CLS430769-1EA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Corning tissue-culture treated culture dishes</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td height="20" style="height:20px;width:139px;">CLS430167</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Trypsin/EDTA Solution (TE)</td>
			<td style="width:128px;">Life technologies</td>
			<td>R001100</td>
			<td style="width:156px;">Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">PIPES dipotassium salt</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td height="20" style="height:20px;width:139px;">108321-27-3&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Calcium acetate hydrate</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td>114460-21-8</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Magnesium acetate tetrahydrate</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td height="20" style="height:20px;width:139px;">M5661&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">L-Glutamic acid potassium salt monohydrate (Potassium glutamate)</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td height="20" style="height:20px;width:139px;">G1501</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">4 mm electroporation cuvettes</td>
			<td style="width:128px;">cell projects</td>
			<td style="width:139px;">EP-104</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">GENE PULSER WITH PULSE CONTROLLER &#38; CAPACITANCE</td>
			<td style="width:128px;">Bio rad</td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Chambered coverglass</td>
			<td style="width:128px;">Thermo scientific</td>
			<td align="right" style="width:139px;">155411</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">24 well, flat bottom</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td height="20" style="height:20px;width:139px;">CLS3524</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Corning 96 well plates</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td height="20" style="height:20px;width:139px;">CLS3367 or CLS390</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">96 well plate fluorescence reader- Infinite 200</td>
			<td style="width:128px;">Tecan</td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Calcium ionophore A23187</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td height="20" style="height:20px;width:139px;">C7522</td>
			<td>Avoid from direct light exposure</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">12-O-tetradecanoyl-13-acetate (TPA)</td>
			<td>Calbiochem</td>
			<td height="20" style="height:20px;width:139px;">P3766</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-DNP monoclonal IgE</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td>D8406&#160;</td>
			<td height="20" style="height:20px;width:156px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DNP-BSA/ DNP-HAS</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td style="width:139px;">A6661</td>
			<td>Avoid from direct light exposure</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Triton-x-100</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td style="width:139px;">T8787</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Confocal fluorescent microscope:</td>
			<td style="width:128px;"></td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Zeiss LSM 510</td>
			<td style="width:128px;"></td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Leica</td>
			<td style="width:128px;">SP5</td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nikon A1 inverted</td>
			<td style="width:128px;"></td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Imaris software</td>
			<td style="width:128px;">BITLANE</td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Microsoft exel or Prism or other analyses software</td>
			<td style="width:128px;"></td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Other reagent:</td>
			<td style="width:128px;"></td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Magnesium Chloride</td>
			<td style="width:128px;">MERK</td>
			<td style="width:139px;">5833</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Sodium chloride</td>
			<td style="width:128px;">MERK</td>
			<td style="width:139px;">6404</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Calcium chloride&#160;</td>
			<td style="width:128px;">MERK</td>
			<td style="width:139px;">2382</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bovine serum albumin&#160;</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td style="width:139px;">A4503</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Glucose</td>
			<td style="width:128px;">BDH Laboratories</td>
			<td style="width:139px;">284515V</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Monosodium phosphate&#160;</td>
			<td style="width:128px;">MERK</td>
			<td style="width:139px;">5345</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Sterile water</td>
			<td style="width:128px;"></td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

investigating mast cell secretory granules; from biosynthesis to exocytosis

Mast Cells (MC) are secretory cells of the immune system that accomplish their physiological and pathological functions by releasing pre-formed and newly synthesized allergic, inflammatory and immunoregulatory mediators. MCs&#8217; mediators affect multiple tissues and organs culminating in allergic and immune responses. The synthesis, storage and release of the MC mediators are highly regulated. The pre-formed mediators are packed in cytoplasmic secretory granules&#160;(SG) that fuse with the plasma membrane and release their content by regulated exocytosis. We present a protocol, based on the co-expression of a gene of interest with a reporter gene that is targeted to the SGs and is released in a regulated fashion alongside the endogenous SG mediators. The protocol enables high resolution four dimensional confocal analyses of the MC SGs and monitoring their timeline from biogenesis to triggered exocytosis. Thus, using this protocol for screening genes of interest for their phenotypic and functional impact allows deciphering the molecular mechanisms that govern the biogenesis and exocytosis of the MC SGs and identifying the regulators involved. Thereby, further insights into the cellular mechanisms that account for MCs function in health and disease should be provided.

Because of the low transfection efficiency of MCs, genetic manipulations are unlikely to leave an impact on readouts of average secretion measured by endogenous SGs mediators. Nevertheless, by establishing complete co-expression of the reporter gene NPY-mRFP and the co-transfected plasmid at the same cells, monitoring of NPY-mRFP results in monitoring exclusively the cell population that expresses the gene of interest. Therefore, the advantage of this assay compared to conventional methods is the ability to selectively m...

Watch this Scientific Journal Video about Investigating Mast Cell Secretory Granules; from Biosynthesis to Exocytosis at JoVE.com

Investigating Mast Cell Secretory Granules; from Biosynthesis to Exocytosis

The goal of the present&#160;protocol was to develop a method that will allow functional genomic analyses of mast cell secretion. The protocol is based on quantitative assessment of the release of a fluorescent reporter gene cotrasfected with the gene of interest and real time&#160;analyses of the secretory granule&#39;s morphology.

Mast Cells (MC) are secretory cells of the immune system that accomplish their physiological and pathological functions by releasing pre-formed and newly ...

investigating-mast-cell-secretory-granules-from-biosynthesis-to

Research

JoVE Journal

Immunology and Infection

13.0K Views.  Sackler Faculty of Medicine, Tel Aviv University. The goal of the present protocol was to develop a method that will allow functional genomic analyses of mast cell secretion. The protocol is based on quantitative assessment of the release of a fluorescent reporter gene cotrasfected with the gene of interest and real time analyses of the secretory granule's morphology.