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 ...

<ol>
	<li>Abonia, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+mast+cells+in+eosinophilic+esophagitis.">Involvement of mast cells in eosinophilic esophagitis.</a> J Allergy Clin Immunol. 126 (1), 140-149 (2010).</li><li>Galli, S. J., Tsai, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mast+cells+in+allergy+and+infection:+versatile+effector+and+regulatory+cells+in+innate+and+adaptive+immunity.">Mast cells in allergy and infection: versatile effector and regulatory cells in innate and adaptive immunity.</a> Eur J Immunol. 40 (7), 1843-1851 (2010).</li><li>Ribatti, D., Crivellato, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+controversial+role+of+mast+cells+in+tumor+growth.">The controversial role of mast cells in tumor growth.</a> International Review of Cell and Molecular Biology. 275, 89-131 (2009).</li><li>Ribatti, D., Crivellato, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mast+cells,+angiogenesis+and+cancer.">Mast cells, angiogenesis and cancer.</a> Adv Exp Med Biol. 716, 270-288 (2011).</li><li>Tsai, M., Grimbaldeston, M., Galli, S. 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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Demonstration+that+human+mast+cells+arise+from+a+progenitor+cell+population+that+is+CD34(+),+c-kit(+),+and+expresses+aminopeptidase+N+(CD13).">Demonstration that human mast cells arise from a progenitor cell population that is CD34(+), c-kit(+), and expresses aminopeptidase N (CD13).</a> Blood. 94 (7), 2333-2342 (1999).</li><li>Metcalfe, D. D., Baram, D., Mekori, Y. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mast+cells.">Mast cells.</a> Physiol Rev. 77 (4), 1033-1079 (1997).</li><li>Gilfillan, A. M., Austin, S. J., Metcalfe, D. 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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mast+cell+proteases+as+protective+and+inflammatory+mediators.">Mast cell proteases as protective and inflammatory mediators.</a> Adv Exp Med Biol. 716, 212-234 (2011).</li><li>Lundequist, A., Pejler, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+implications+of+preformed+mast+cell+mediators.">Biological implications of preformed mast cell mediators.</a> Cell Mol Life Sci. 68 (6), 965-975 (2011).</li><li>Metz, M., Maurer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mast+cells--key+effector+cells+in+immune+responses.">Mast cells--key effector cells in immune responses.</a> Trends Immunol. 28 (5), 234-241 (2007).</li><li>Gordon, J. R., Burd, P. 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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.

<table border="0" cellpadding="0" cellspacing="0" width="844">
	<colgroup>
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		<col />
		<col />
		<col />
	</colgroup>
	<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>
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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...

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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 visualizações.  Sackler Faculty of Medicine, Tel Aviv University.  O objectivo do presente protocolo foi o de desenvolver um método que permita análises genómicas funcionais da secreção de mastócitos. O protocolo baseia-se na avaliação quantitativa da libertação de um gene repórter fluorescente cotrasfected com o gene de interesse e análises em tempo real da morfologia do grânulo secretor. 