ZG es apoyada por las subvenciones del Programa de I-CORE de la Fundación de Ciencias de Israel (Grant No. 41/11), la Fundación Abisch-Frenkel, la rosales Trust, la Fundación de Investigación de Cáncer de Israel (ICRF - Premio al Desarrollo de Carreras de Investigación) y la fundación PREOCUPACIÓN. ZGF es apoyado por becas de la Fundación de Investigación del Cáncer de Israel (ICRF - Premio al Desarrollo de Carreras de Investigación), Jefe Científico del Ministerio de Salud de Israel y la Asociación del Pulmón Israel.

<ol>
	<li>Tangye, S. G., Brink, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+helping+hand+from+neutrophils+in+T+cell-independent+antibody+responses.">A helping hand from neutrophils in T cell-independent antibody responses.</a> Nat Immunol. 13, 111-113 (2012).</li><li>Mantovani, A., Cassatella, M. A., Costantini, C., Jaillon, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophils+in+the+activation+and+regulation+of+innate+and+adaptive+immunity.">Neutrophils in the activation and regulation of innate and adaptive immunity.</a> Nat Rev Immunol. 11, 519-531 (2011).</li><li>Pruijt, J. F., 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=Neutrophils+are+indispensable+for+hematopoietic+stem+cell+mobilization+induced+by+interleukin-8+in+mice.">Neutrophils are indispensable for hematopoietic stem cell mobilization induced by interleukin-8 in mice.</a> Proc Natl Acad Sci U S A. 99, 6228-6233 (2002).</li><li>Tecchio, C., Cassatella, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil-derived+cytokines+involved+in+physiological+and+pathological+angiogenesis.">Neutrophil-derived cytokines involved in physiological and pathological angiogenesis.</a> Chem Immunol Allergy. 99, 123-137 (2014).</li><li>Liu, M., 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=Formylpeptide+receptors+mediate+rapid+neutrophil+mobilization+to+accelerate+wound+healing.">Formylpeptide receptors mediate rapid neutrophil mobilization to accelerate wound healing.</a> PloS one. 9, e90613 (2014).</li><li>Granot, Z., 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=Tumor+entrained+neutrophils+inhibit+seeding+in+the+premetastatic+lung.">Tumor entrained neutrophils inhibit seeding in the premetastatic lung.</a> Cancer Cell. 20, 300-314 (2011).</li><li>Sionov, R. V., Fridlender, Z. G., Granot, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Multifaceted+Roles+Neutrophils+Play+in+the+Tumor+Microenvironment.">The Multifaceted Roles Neutrophils Play in the Tumor Microenvironment.</a> Cancer Microenviron. , (2014).</li><li>Borregaard, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophils,+from+marrow+to+microbes.">Neutrophils, from marrow to microbes.</a> Immunity. 33, 657-670 (2010).</li><li>Yamashiro, 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=Phenotypic+and+functional+change+of+cytokine-activated+neutrophils:+inflammatory+neutrophils+are+heterogeneous+and+enhance+adaptive+immune+responses.">Phenotypic and functional change of cytokine-activated neutrophils: inflammatory neutrophils are heterogeneous and enhance adaptive immune responses.</a> J Leukoc Biol. 69, 698-704 (2001).</li><li>Futosi, K., Fodor, S., Mocsai, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+cell+surface+receptors+and+their+intracellular+signal+transduction+pathways.">Neutrophil cell surface receptors and their intracellular signal transduction pathways.</a> Int Immunopharmacol. 17, 638-650 (2013).</li><li>Scapini, P., Cassatella, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Social+networking+of+human+neutrophils+within+the+immune+system.">Social networking of human neutrophils within the immune system.</a> Blood. 124, 710-719 (2014).</li><li>Fridlender, Z. G., 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=Transcriptomic+analysis+comparing+tumor-associated+neutrophils+with+granulocytic+myeloid-derived+suppressor+cells+and+normal+neutrophils.">Transcriptomic analysis comparing tumor-associated neutrophils with granulocytic myeloid-derived suppressor cells and normal neutrophils.</a> PloS one. 7, e31524 (2012).</li><li>Fridlender, Z. G., 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=Polarization+of+tumor-associated+neutrophil+phenotype+by+TGF-beta:+'N1'+versus+'N2.">Polarization of tumor-associated neutrophil phenotype by TGF-beta: 'N1' versus 'N2.</a> TAN. Cancer Cell. 16, 183-194 (2009).</li><li>Talmadge, J. E., Gabrilovich, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=History+of+myeloid-derived+suppressor+cells.">History of myeloid-derived suppressor cells.</a> Nat Rev Cancer. 13, 739-752 (2013).</li><li>Youn, J. I., Nagaraj, S., Collazo, M., Gabrilovich, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subsets+of+myeloid-derived+suppressor+cells+in+tumor-bearing+mice.">Subsets of myeloid-derived suppressor cells in tumor-bearing mice.</a> J Immunol. 181, 5791-5802 (2008).</li><li>Choi, J., 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=CD15+/CD16low+human+granulocytes+from+terminal+cancer+patients:+granulocytic+myeloid-derived+suppressor+cells+that+have+suppressive+function.">CD15+/CD16low human granulocytes from terminal cancer patients: granulocytic myeloid-derived suppressor cells that have suppressive function.</a> Tumour Biol. 33, 121-129 (2012).</li><li>Brandau, 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=Myeloid-derived+suppressor+cells+in+the+peripheral+blood+of+cancer+patients+contain+a+subset+of+immature+neutrophils+with+impaired+migratory+properties.">Myeloid-derived suppressor cells in the peripheral blood of cancer patients contain a subset of immature neutrophils with impaired migratory properties.</a> J Leukoc Biol. 89, 311-317 (2011).</li><li>Pillay, J., Tak, T., Kamp, V. M., Koenderman, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+suppression+by+neutrophils+and+granulocytic+myeloid-derived+suppressor+cells:+similarities+and+differences.">Immune suppression by neutrophils and granulocytic myeloid-derived suppressor cells: similarities and differences.</a> Cell Mol Life Sci. 70, 3813-3827 (2013).</li><li>Lopez-Lago, M. A., 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=Neutrophil+chemokines+secreted+by+tumor+cells+mount+a+lung+antimetastatic+response+during+renal+cell+carcinoma+progression.">Neutrophil chemokines secreted by tumor cells mount a lung antimetastatic response during renal cell carcinoma progression.</a> Oncogene. 32, 1752-1760 (2013).</li><li>Boxio, R., Bossenmeyer-Pourie, C., Steinckwich, N., Dournon, C., Nusse, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+bone+marrow+contains+large+numbers+of+functionally+competent+neutrophils.">Mouse bone marrow contains large numbers of functionally competent neutrophils.</a> J Leukoc Biol. 75, 604-611 (2004).</li><li>Sagiv, J., 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=Phenotypic+Diversity+and+Plasticity+in+Circulating+Neutrophil+Subpopulations+in+Cancer.">Phenotypic Diversity and Plasticity in Circulating Neutrophil Subpopulations in Cancer.</a> Cell Reports. , (2015).</li></ol>

The authors have nothing to disclose.

Los neutrófilos son las más abundantes de todas las células blancas de la sangre y son los primeros en responder en casos de infección y la inflamación. Como tales, son muy sensibles a las señales externas y se activan fácilmente. Además, los neutrófilos tienen una vida media muy corta y una rápida rotación. En conjunto, estas características plantean varias dificultades en el trabajo con los neutrófilos, de tal manera que se requieren estrategias experimentales únicas. Por ejemplo, hay varias estrategias ...

Los neutrófilos se caracterizaron inicialmente como las células inmunes innatas que sirven como primera línea de defensa contra los microorganismos invasores. Hoy en día se sabe que los neutrófilos han más funciones de gran alcance, estar involucrado en el montaje de la respuesta inmune adaptativa contra antígenos extraños 1,2, regulación de la hematopoyesis 3, 4 angiogénesis y cicatrización de heridas 5. Además, los neutrófilos pueden afectar el crecimiento del tumor y la progresión metastásica en virtud de sus actividades pro- y anti-tumorales 6,7. Los neutrófilos se caracterizan por un núcleo segmentado polimórfica (por lo tanto denominado polimorfonucleares (PMN) leucocitos) y contienen al menos tres subclases distintas de gránulos, así como vesículas secretoras 8 (Figura 1A-C). Los neutrófilos poseen alta capacidad fagocítica y de alta actividad de la oxidasa NADPH crítico para la eliminación microbiana, y secretan una amplia gama de quimioquinas importantes por lotracción de los neutrófilos adicionales y otras células inmunes en el sitio de la inflamación 8,9. Los neutrófilos se caracterizan por la expresión de una gran cantidad de receptores de la superficie, incluyendo los receptores de tipo Toll (TLRs), lectina tipo C receptores (CLR), receptor del complemento 3 (CD11b / CD18) y otras moléculas de adhesión (por ejemplo, L-selectina, LFA-1, VLA-4 y carcinoembrionario relacionados antígeno-molécula de adhesión celular 3 (CEACAM3 / CD66b)), receptores de quimiocinas (por ejemplo, CXCR1, CXCR2, CCR1, CCR2), los receptores quimioatrayentes (por ejemplo, PAFR, LTB 4 y R C5aR) , receptores de citoquinas (por ejemplo, G-CSFR, IL-1R, IL-4R, IL-12R, IL-18R, TNFR), formil-receptores de péptidos (por ejemplo, FPR1-3), y los receptores de Fc (por ejemplo, CD16 (Fc? RIII ), CD32 (RII) y CD64 (Fc? RI) 10. En los ratones, los neutrófilos suelen identificarse como CD11b + Ly6G +, mientras que los neutrófilos humanos se identifican utilizando los marcadores de leucocitos CD11b, CD15, CD16 y CD66b. También es generalmente aceptado para teñir para la mieloperoxidasa proteínas granulares (MPO) y la elastasa de neutrófilos (NE) para la detección de los neutrófilos en los tejidos. Aún no está claro si las diversas funciones de los neutrófilos están mediados por la misma célula o por sub-poblaciones de células distintas. La acumulación de datos sugieren la presencia de una población heterogénea de neutrófilos que exhibe un alto grado de plasticidad afectados por estímulos proinflamatorios y el microambiente 11,12. Fridlender et al. 13 groseramente han dividido a los neutrófilos en el cáncer en dos grandes grupos de población denominado N1 con propiedades antitumorales y N2 con propiedades pro-tumorales. En el cáncer, así como en la inflamación crónica, existe una sub-población adicional compuesta de células granulocíticas mieloides derivadas de supresores (G-MDSCs) que suprimen las respuestas de células T 14. G-MDSCs se considera que son las células mieloides inmaduras se caracterizan por un CD11b + Ly6Cbajo hi fenotipo Ly6G en ratones 15, mientras que con un CD15 + / CD16 baja fenotipo en humanos 16. G-MDSCs expresan niveles más altos de arginasa y la mieloperoxidasa, mientras que los niveles más bajos de citocinas y quimiocinas que los neutrófilos circulantes normales. Son menos de fagocitosis y migratoria, pero producen mayores niveles de ROS 15,17,18. En el presente artículo vamos a describir algunas metodologías básicas para el aislamiento y la caracterización de los neutrófilos con propiedades antitumorales. Mientras que los neutrófilos constituyen la población más grande de todas las células blancas de la sangre en la circulación humana (45 - 70%; 1800 - 6000 / l), en ratones, en condiciones normales, son más bien escasa (10 - 15%; 300-500 / l ). El recuento de neutrófilos aumenta de manera constante sobre la inflamación y en ocasiones en el cáncer, lo que representa un estado de inflamación crónica 7. Los neutrófilos se desarrollan a partir multipotentes precursor mieloide común (CMP) cells en la médula ósea, a través de un proceso de diferenciación que pasan las etapas de mieloblastos (MB), promielocitos (PM), mielocitos (MC), metamielocitos (MM) y células de la banda (BC) 8. Los neutrófilos, post-mitóticas maduras pueden permanecer dentro de la médula ósea de 4-7 días antes de que se liberan a la circulación 8. Facturación de neutrófilos en la sangre suele ser rápida con una vida media promedio de 6 - 12 horas, que puede ser prolongado en condiciones inflamatorias. Neutrófilos no estimulados han limitado la actividad anti-tumorigénicos, una característica que puede ser adquirida mediante la exposición de los neutrófilos ingenuos a las quimiocinas IL-8 (CXCL2), CCL2, CCL5 y CXCL5 6,19 o artificialmente, al exponerlos a la forbol éster de forbol 12 -myristate 13-acetato (PMA) 6. La corta vida media de los neutrófilos en la sangre junto con el bajo número de neutrófilos (~ 3-5 x 10 5) alcanzado entre el 1 ml de sangre de un inocente 6-8 semanas de edad ratón, han hechodifícil de explorar la función de los neutrófilos circulantes ratón in vitro. Para superar esta dificultad, se han utilizado otras fuentes. Por ejemplo, un gran número de neutrófilos pueden ser obtenidas de la médula ósea 20 o el peritoneo tras la inducción de la inflamación estéril (por ejemplo, después de la inyección intraperitoneal de caldo de tioglicolato o Zymosan A). Cabe señalar que los neutrófilos obtenidos a partir de la cavidad peritoneal no ejercen ninguna actividad anti-tumorigénico (observación no publicada). . Granot et al 6 observó que los ratones BALB / c inoculados ortotópicamente con el ratón 4T1 línea celular de carcinoma de mama desarrollar neutrofilia que agrava con la progresión tumoral 6 (Figura 2A), de tal manera que 20 a 40000000 neutrófilos de la sangre pueden ser fácilmente aislados de 1 ml de sangre 3 - 4 weeks post-inoculación del tumor. Estos neutrófilos han adquirido las actividades antitumorales, y en consecuencia han sido coineutrófilos nidos tumorales arrastradas (RTE), a fin de distinguirlos de los neutrófilos ingenuos 6 (Figura 2B). Mientras que los neutrófilos de alta densidad (HDN, la Figura 1A) son altamente anti-tumorigénicos, los neutrófilos de baja densidad (LDN, Figura 1B) generados en el contexto de cáncer no son 21. Además, los neutrófilos de alta densidad de la médula ósea y el bazo de ratones portadores de tumores tienen actividad anti-tumor (datos no publicados). Cabe señalar que con la progresión del tumor del bazo se vuelve gradualmente agrandada (esplenomegalia), con cantidades crecientes de neutrófilos. Cabe señalar que la RTE se generan también en otros modelos de cáncer incluyendo tanto espontáneas (MMTV-PYMT y MMTV-Wnt1 tumores mamarios y tumores de pulmón impulsados ​​k-Ras) y se inyectaron (AT-3 (MMTV-PYMT) y carcinoma de mama E0771 células, células de carcinoma de pulmón de Lewis LLC y células de melanoma B16-F10). Sin embargo, la medida de la movilización de neutrófilos en estos tumo modelos es mucho menor que los ratones inoculados-4T1, alcanzando 5-10 x 10 6 neutrófilos en 1 ml de sangre después de 3 semanas.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>CELL LINES</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse 4T1 breast carcinoma cells</td>
			<td>ADCC</td>
			<td>CRL-2539</td>
			<td>Growth medium: DMEM + 10 % heat-inactivated FBS</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PLASTIC WARES AND EQUIPMENTS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>24-well Tissue Culture Plate&#160;</td>
			<td>Falcon</td>
			<td>353047</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>100 mm Tissue Culture Plate&#160;</td>
			<td>Corning</td>
			<td>430167</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>25 cm2 Tissue Culture Flask</td>
			<td>Nunc</td>
			<td>156340</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>90 mm Bacterial Grade Culture Dish&#160;</td>
			<td>Miniplast, Ein Shemer, Israel</td>
			<td>20090-01-017</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>15 ml Sterile Conical Centrifuge Tube&#160;</td>
			<td>Miniplast, Ein Shemer, Israel</td>
			<td>835015-40-111</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>50 ml Sterile Conical Centrifuge Tube&#160;</td>
			<td>Miniplast, Ein Shemer, Israel</td>
			<td>835050-21-111</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>Falcon 12x75 mm Round-Bottom Polystyrene Tube&#160;</td>
			<td>Becton Dickinson</td>
			<td>352058</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>Millicell 24 Migration Plate with a pore size of 5&#956;m&#160;</td>
			<td>Merck Millipore</td>
			<td>PSMT010R1</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>White 96-Flat-Bottom Well Plate&#160;</td>
			<td>Costar</td>
			<td>3917</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>Cell Strainer (40 mm)&#160;</td>
			<td>BD Falcon</td>
			<td>352340</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>20G 1.5&#34; Needle</td>
			<td>BD Microlance 3&#160;</td>
			<td>301300</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>23G 1&#34; Needle&#160;</td>
			<td>BD Microlance 4</td>
			<td>300800</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>25Gx5/8&#34; Needle&#160;</td>
			<td>BD Microlance 5</td>
			<td>300600</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>0.3 ml Syringe with a 30Gx8mm Needle</td>
			<td>BD Micro-Fine Plus Demi</td>
			<td>320829</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>9 mm Clips&#160;</td>
			<td>BD, AutoClip&#160;</td>
			<td>427631</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>EasySep Magnet&#160;</td>
			<td>STEMCELL Technologies</td>
			<td>18000</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS LS Separation Column&#160;</td>
			<td>Miltenyi Biotech</td>
			<td>130-042-201</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>MidiMACS Separator Magnet</td>
			<td>Miltenyi Biotech</td>
			<td>130-042-302</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS MultiStand</td>
			<td>Miltenyi Biotech</td>
			<td>130-042-303</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Glass Slide&#160;</td>
			<td>Menzel-Gl&#228;ser Superfrost&#160; Plus Thermo</td>
			<td>J1800AMNZ</td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital Shaker&#160;</td>
			<td>Sky line, ELMI</td>
			<td>S-3.02.10L</td>
			<td></td>
		</tr>
		<tr>
			<td>Plate Reader&#160;</td>
			<td>TECAN</td>
			<td>InfiniteF200Pro</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>POWDER</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA), fraction V</td>
			<td>Sigma</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>Bromodeoxyuridine (BrdU)&#160;</td>
			<td>BD Pharmingen</td>
			<td>550891</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>CFSE (5-(and 6-)-Carboxyfluorescein diacetate, succinimidyl ester)</td>
			<td>Molecular Probes</td>
			<td>C1157</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextran T500</td>
			<td>Sigma</td>
			<td>31392</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin sodium salt from porcine intestinal mucosa&#160;</td>
			<td>Sigma</td>
			<td>H3149</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium azide (NaN3)</td>
			<td>Sigma</td>
			<td>S8032</td>
			<td>Highly toxic, handle with care</td>
		</tr>
		<tr>
			<td>Thioglycollate powder&#160;</td>
			<td>Difco</td>
			<td>225650</td>
			<td></td>
		</tr>
		<tr>
			<td>Zymosan A</td>
			<td>Sigma</td>
			<td>Z4250</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MEDIA AND SUPPLEMENTS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified Eagle medium (DMEM)</td>
			<td>Sigma</td>
			<td>D5796</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>Opti-MEM&#174; I reduced serum medium&#160;</td>
			<td>Life Technologies</td>
			<td>31985062</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>Roswell Park Memorial Institute (RPMI)-1640 medium</td>
			<td>Sigma</td>
			<td>R8758</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>Foetal bovine serum (FBS), heat-inactivated</td>
			<td>Sigma</td>
			<td>F9665</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Biological Industries, Beth HaEmek, Israel</td>
			<td>03-020-1A</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Biological Industries, Beth HaEmek, Israel</td>
			<td>03-042-1B</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin x1000 solution</td>
			<td>Biological Industries, Beth HaEmek, Israel</td>
			<td>03-031-5</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS) without Mg2+ and Ca2+&#160;</td>
			<td>Biological Industries, Beth HaEmek, Israel</td>
			<td>02-023-1</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>PBSx10 without Ca2+ and Mg2+&#160;</td>
			<td>Biological Industries, Beth HaEmek, Israel</td>
			<td>02-023-5A</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>HPLC grade water&#160;</td>
			<td>J.T. Baker</td>
			<td>4218-03</td>
			<td>Autoclave</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SOLUTIONS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ACK &#8211; Ammonium-Chloride-Potassium</td>
			<td>Life Technologies&#160;</td>
			<td>A10492-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Bromodeoxyuridine (BrdU) solution (10 mg/ml) in PBS</td>
			<td></td>
			<td></td>
			<td>Dissolve 10 mg of BrdU in 1 ml PBS and sterile filter.</td>
		</tr>
		<tr>
			<td>CFSE, 5 mM in DMSO</td>
			<td></td>
			<td></td>
			<td>Dissolve 2.8 mg of CFSE in 1 ml DMSO. Divide into 10 ml aliquots in sterile 200 ml tubes and store in the dark at -20oC.</td>
		</tr>
		<tr>
			<td>Eosin Y solution</td>
			<td>Sigma</td>
			<td>HT110-2-32</td>
			<td></td>
		</tr>
		<tr>
			<td>Hanks&#39; balanced salt solution</td>
			<td>Biological Industries, Beth HaEmek, Israel</td>
			<td>02-016-1A</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>Heparin, 20 mg/ml in PBS</td>
			<td></td>
			<td></td>
			<td>Dissolve 100 mg Heparin in 5 ml sterile PBS, and sterile filter through a 0.2 mm filter.&#160;</td>
		</tr>
		<tr>
			<td>Histopaque-1119&#160;</td>
			<td>Sigma</td>
			<td>11191</td>
			<td>Sterile filter through a 0.2 mm filter.</td>
		</tr>
		<tr>
			<td>Histopaque-1077&#160;</td>
			<td>Sigma</td>
			<td>10771</td>
			<td>Sterile filter through a 0.2 mm filter.</td>
		</tr>
		<tr>
			<td>&#160;Luciferase cell culture lysis buffer x5</td>
			<td>Promega</td>
			<td>E153A</td>
			<td>Dilute 1:5 in sterile water just before use.</td>
		</tr>
		<tr>
			<td>Luciferase assay solution</td>
			<td>Promega</td>
			<td>E1501</td>
			<td>Contains luciferase assay substrate powder (E151A) and luciferase assay buffer (E152A)</td>
		</tr>
		<tr>
			<td>Mayer&#39;s Hematoxylin solution&#160;</td>
			<td>Sigma</td>
			<td>MHS-32</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS+0.5% BSA</td>
			<td></td>
			<td></td>
			<td>Dissolve 2.5g BSA in 500 ml PBS, and sterile filter through a 0.2 mm filter.</td>
		</tr>
		<tr>
			<td>PBS+1% BSA</td>
			<td></td>
			<td></td>
			<td>Dissolve 1g BSA in 100 ml PBS, and sterile filter through a 0.2 mm filter.</td>
		</tr>
		<tr>
			<td>5x PBS with 2.5% BSA</td>
			<td></td>
			<td></td>
			<td>&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Dissolve 12.5g BSA in a mixture of 250 ml sterile HPLC-grade water&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; and 250 ml PBSx10, and sterile filter through a 0.2 mm filter.</td>
		</tr>
		<tr>
			<td>PBS containing 0.5% BSA and 2 mM EDTA</td>
			<td></td>
			<td></td>
			<td>&#160;&#160;&#160;&#160;&#160;&#160;&#160; Dissolve 250 mg BSA in 50 ml sterile PBS&#160; and add 200 ml of 0.5M EDTA pH 8.0, sterile filter through a 0.2 mm filter.</td>
		</tr>
		<tr>
			<td>FACS buffer (PBS containing 0.5% BSA, 2 mM EDTA and 0.02% NaN3)</td>
			<td></td>
			<td></td>
			<td>&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Dissolve 250 mg BSA in 50 ml sterile PBS&#160; and add 200 ml of 0.5M EDTA pH 8.0 and 500 ml of 2% NaN3, sterile filter through a 0.2 mm filter.</td>
		</tr>
		<tr>
			<td>Saline (0.9% NaCl)</td>
			<td></td>
			<td></td>
			<td>Dissolve 9 g NaCl in 1000 ml ddw, autoclave</td>
		</tr>
		<tr>
			<td>0.2% NaCl solution</td>
			<td></td>
			<td></td>
			<td>Dissolve 2 g NaCl in 1000 ml ddw, autoclave</td>
		</tr>
		<tr>
			<td>1.6% NaCl solution</td>
			<td></td>
			<td></td>
			<td>Dissolve 16 g NaCl in 1000 ml ddw, autoclave</td>
		</tr>
		<tr>
			<td>2 % Sodium azide</td>
			<td></td>
			<td></td>
			<td>Dissolve 1g sodium azide in 50 ml sterile ddw, keep at 4oC. Highly toxic.</td>
		</tr>
		<tr>
			<td>3% Thioglycollate solution</td>
			<td></td>
			<td></td>
			<td>&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Dissolve 3 g of thioglycollate powder in 100 ml ddw.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Boil until solution becomes yellow and autoclave.</td>
		</tr>
		<tr>
			<td>Trypan blue solution (0.4%)</td>
			<td>Sigma</td>
			<td>T8154</td>
			<td>Dilute 1:10 in PBS to get a 0.04% solution.</td>
		</tr>
		<tr>
			<td>Trypsin solution B&#160;</td>
			<td>Biological Industries, Beth HaEmek, Israel</td>
			<td>03-046-1</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>1 mg/ml Zymosan A</td>
			<td></td>
			<td></td>
			<td>&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Resuspend 1 mg Zymosan A&#160; in 1 ml sterile PBS in an Eppendorf tube.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Vortex vigorously and incubate the tube at 37 oC for 30 min. Do not autoclave.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Prepare the solution freshly before use.&#160;</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>KITS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EasySep PE selection kit</td>
			<td>STEMCELL Technologies</td>
			<td>18557</td>
			<td></td>
		</tr>
		<tr>
			<td>EasySep PE selection cocktail&#160;</td>
			<td>STEMCELL Technologies</td>
			<td>18151</td>
			<td></td>
		</tr>
		<tr>
			<td>the EasySep magnetic nanoparticles&#160;</td>
			<td>STEMCELL Technologies</td>
			<td>18150</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Ly6G mouse MicroBead Kit</td>
			<td>Miltenyi Biotec</td>
			<td>130-092-332</td>
			<td></td>
		</tr>
		<tr>
			<td>EasySep Mouse Neutrophil Enrichment Kit</td>
			<td>STEMCELL Technologies</td>
			<td>19762</td>
			<td></td>
		</tr>
		<tr>
			<td>EasySep Human Neutrophil Enrichment Kit</td>
			<td>STEMCELL Technologies</td>
			<td>19257</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC BrdU flow kit</td>
			<td>BD Pharmingen&#160;</td>
			<td>559619</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS Neutrophil isolation kit</td>
			<td>Miltenyi Biotec</td>
			<td>130-097-658</td>
			<td></td>
		</tr>
		<tr>
			<td>Phagocytosis Assay Kit&#160;</td>
			<td>Cayman Chemical Company&#160;</td>
			<td>500290</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ANTIBODIES</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FcR blocking antibody&#160;</td>
			<td>Biolegend</td>
			<td>101302</td>
			<td></td>
		</tr>
		<tr>
			<td>Purified rat anti-Ly6G antibody&#160;</td>
			<td>BD Pharmingen&#160;</td>
			<td>551459</td>
			<td>Clone 1A8</td>
		</tr>
		<tr>
			<td>PE-conjugated rat anti-mouse Ly6G antibody&#160;</td>
			<td>Biolegend</td>
			<td>127608</td>
			<td>Clone 1A8</td>
		</tr>
		<tr>
			<td>FITC-conjugated rat anti-mouse Ly6G</td>
			<td>BD Pharmingen&#160;</td>
			<td>551460</td>
			<td>Clone 1A8</td>
		</tr>
		<tr>
			<td>PerCP-Cy5.5 rat anti-mouse Ly6G&#160;</td>
			<td>TONBO Biosciences</td>
			<td>65-1276</td>
			<td>Clone 1A8</td>
		</tr>
		<tr>
			<td>violetFluor 450-conjugated rat anti-mouse Ly6G&#160;</td>
			<td>TONBO Biosciences</td>
			<td>75-1276</td>
			<td>Clone 1A8</td>
		</tr>
		<tr>
			<td>FITC-conjugated rat anti-mouse CD11b</td>
			<td>BD Pharmingen&#160;</td>
			<td>553310</td>
			<td>Clone M1/70</td>
		</tr>
		<tr>
			<td>FITC-conjugated rat anti-mouse Ly-6G and Ly-6C (GR-1)</td>
			<td>BD Pharmingen&#160;</td>
			<td>553127</td>
			<td>Clone RB6-8C5</td>
		</tr>
		<tr>
			<td>PE-conjugated rat anti-mouse CD45</td>
			<td>BD Pharmingen&#160;</td>
			<td>553081</td>
			<td>Clone 30-F11</td>
		</tr>
		<tr>
			<td>FITC-conjugated rat anti-mouse F4/80&#160;</td>
			<td>Abcam</td>
			<td>ab60343</td>
			<td>Clone BM8</td>
		</tr>
		<tr>
			<td>FITC-conjugated mouse anti-human CD66b&#160;</td>
			<td>Biolegend</td>
			<td>305103</td>
			<td>Clone G10F5</td>
		</tr>
		<tr>
			<td>Purified rat isotype control antibody (IgG2a, k)&#160;</td>
			<td>BD Pharmingen&#160;</td>
			<td>553927</td>
			<td>Clone R35-95</td>
		</tr>
		<tr>
			<td>LEAF purified Armenian hamster anti-mouse CD3e antibody</td>
			<td>BioLegend</td>
			<td>100314</td>
			<td>Clone 145-2C11</td>
		</tr>
	</tbody>
</table>

isolation and characterization of neutrophils with anti-tumor properties

Los neutrófilos, la más abundante de todas las células blancas de la sangre en la circulación humana, juegan un papel importante en la defensa del huésped contra los microorganismos invasores. Además, los neutrófilos desempeñan un papel central en la vigilancia inmune de las células tumorales. Tienen la capacidad de reconocer las células tumorales e inducir la muerte de células tumorales, ya sea a través de un mecanismo dependiente del contacto celular que implica peróxido de hidrógeno o a través de citotoxicidad mediada por células dependiente de anticuerpos (ADCC). Los neutrófilos con actividad anti-tumor se pueden aislar a partir de sangre periférica de pacientes con cáncer y de ratones portadores de tumores. Estos neutrófilos se denominan neutrófilos tumorales arrastradas (diez) para distinguirlos de los neutrófilos de sujetos sanos o ratones naïve que no muestran actividad citotóxica significativa tumor. En comparación con otras células blancas de la sangre, los neutrófilos muestran diferente flotabilidad por lo que es factible obtener a&gt; 98% de la población de neutrófilos puro cuando se somete a un gradiente de densidad. Sin embargo, ademása la población de neutrófilos de alta densidad normal (HDN), en pacientes con cáncer, en ratones portadores de tumores, así como en condiciones inflamatorias crónicas, las poblaciones de neutrófilos de baja densidad distintas (LDN) aparecen en la circulación. LDN co-purifica con la fracción mononuclear y se puede separar a partir de células mononucleares utilizando cualquiera de las estrategias de selección positivos o negativos. Una vez que la pureza de los neutrófilos aislados se determina por citometría de flujo, pueden ser utilizados para in vitro y en ensayos funcionales in vivo. Se describen técnicas para el seguimiento de la actividad anti-tumor de los neutrófilos, su capacidad para migrar y para producir especies reactivas del oxígeno, así como la supervisión de su capacidad fagocítica ex vivo. Describimos más técnicas para etiquetar los neutrófilos para el seguimiento in vivo, y para determinar su capacidad anti-metastásico in vivo. Todas estas técnicas son esenciales para la comprensión de cómo obtener y caracterizar los neutrófilos con anti-tumorfunción.

En un estudio reciente se identificó una función anti-metastásica de neutrófilos 6. Los neutrófilos de ratones portadores de tumores adquirir un fenotipo citotóxico y tienen la capacidad para matar las células tumorales 6. Esto está en contraste con los neutrófilos de ratones naïve que no tienen efecto anti-tumoral significativa 6. Varias de las técnicas descritas en la Sección de Protocolo se han utilizado para el estudio de la función de neutrófilos anti-tumor in vitr...

Watch this Scientific Journal Video about Isolation and Characterization of Neutrophils with Anti-Tumor Properties at JoVE.com

Isolation and Characterization of Neutrophils with Anti-Tumor Properties

Neutrophils play an important role not only in host defense against invading microorganisms, but are also involved in the immune surveillance of tumor cells. Here, we describe techniques related to the isolation of neutrophils with anti-tumor properties and methods for monitoring anti-tumor neutrophil function in vitro and in vivo.

Aislamiento y caracterización de los neutrófilos con propiedades antitumorales

Neutrophils, the most abundant of all white blood cells in the human circulation, play an important role in the host defense against invading ...

isolation-characterization-neutrophils-with-anti-tumor

Research

JoVE Journal

Immunology and Infection

24.6K vistas.  Hebrew University Medical School. Neutrophils play an important role not only in host defense against invading microorganisms, but are also involved in the immune surveillance of tumor cells. Here, we describe techniques related to the isolation of neutrophils with anti-tumor properties and methods for monitoring anti-tumor neutrophil function in vitro and in vivo. 

Video: Isolation and Characterization of Neutrophils with Anti-Tumor Properties

Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the generation of extreme population diversity that facilitates virus adaptation and evolution. Increasing evidence shows that the intrinsic error rates, and the resulting mutation frequencies, of RNA viruses can be modulated by subtle amino acid changes to the viral polymerase. Although biochemical assays exist for some viral RNA polymerases that permit quantitative measure of incorporation fidelity, here we describe a simple method of measuring mutation frequencies of RNA viruses that has proven to be as accurate as biochemical approaches in identifying fidelity altering mutations. The approach uses conventional virological and sequencing techniques that can be performed in most biology laboratories. Based on our experience with a number of different viruses, we have identified the key steps that must be optimized to increase the likelihood of isolating fidelity variants and generating data of statistical significance. The isolation and characterization of fidelity altering mutations can provide new insights into polymerase structure and function1-3. Furthermore, these fidelity variants can be useful tools in characterizing mechanisms of virus adaptation and evolution4-7.

The present article describes the steps required to isolate and characterize RNA polymerase fidelity variants of RNA viruses and how to use mutation frequency data to confirm fidelity changes in tissue culture.

El aislamiento de las variantes de los virus de ARN de fidelidad y caracterización de la frecuencia de mutación de virus

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the ...

Investigación

Inmunología e Infección

Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and food antigens while concomitantly remaining poised to mount inflammatory responses toward invasive pathogens1,2. Antigen presenting cells, particularly DCs and macrophages, play critical roles in maintaining intestinal immune homeostasis via their ability to sense and appropriately respond to the microbiota3-14. Efficient isolation of intestinal DCs and macrophages is a critical step in characterizing the phenotype and function of these cells. While many effective methods of isolating intestinal immune cells, including DCs and macrophages, have been described6,10,15-24, many rely upon long digestions times that may negatively influence cell surface antigen expression, cell viability, and/or cell yield. Here, we detail a methodology for the rapid isolation of large numbers of viable, intestinal DCs and macrophages. Phenotypic characterization of intestinal DCs and macrophages is carried out by directly staining isolated intestinal cells with specific fluorescence-labeled monoclonal antibodies for multi-color flow cytometric analysis. Furthermore, highly pure DC and macrophage populations are isolated for functional studies utilizing CD11c and CD11b magnetic-activated cell sorting beads followed by cell sorting.

Here, we detail a methodology for the rapid isolation of mouse intestinal dendritic cells (DCs) and macrophages. Phenotypic characterization of intestinal DCs and macrophages is performed using multi-color flow cytometric analysis while magnetic bead enrichment followed by cell sorting is used to yield highly pure populations for functional studies.

Aislamiento y caracterización de las células dendríticas y los macrófagos del intestino delgado de ratón

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and ...

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing the mucosal surface of the nasopharynx, where it can reside, multiply and eventually overcome host defences to invade to other tissues of the host. Establishment of an infection in the normally lower respiratory tract results in pneumonia. Alternatively, the bacteria can disseminate into the bloodstream causing bacteraemia, which is associated with high mortality rates2, or else lead directly to the development of pneumococcal meningitis. Understanding the kinetics of, and immune responses to, nasopharyngeal colonization is an important aspect of S. pneumoniae infection models.
Our mouse model of intranasal colonization is adapted from human models3 and has been used by multiple research groups in the study of host-pathogen responses in the nasopharynx4-7. In the first part of the model, we use a clinical isolate of S. pneumoniae to establish a self-limiting bacterial colonization that is similar to carriage events in human adults. The procedure detailed herein involves preparation of a bacterial inoculum, followed by the establishment of a colonization event through delivery of the inoculum via an intranasal route of administration. Resident macrophages are the predominant cell type in the nasopharynx during the steady state. Typically, there are few lymphocytes present in uninfected mice8, however mucosal colonization will lead to low- to high-grade inflammation (depending on the virulence of the bacterial species and strain) that will result in an immune response and the subsequent recruitment of host immune cells. These cells can be isolated by a lavage of the tracheal contents through the nares, and correlated to the density of colonization bacteria to better understand the kinetics of the infection.

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Colonization of the murine nasopharynx with Streptococcus pneumoniae and the subsequent extraction of adherent or recruited cells is described. This technique involves flushing the nasopharynx and collection of the fluid through the nares and is adaptable for various readouts, including differential cell quantification and analysis of mRNA expression in situ.

Caracterización De Las Respuestas Inflamatorias Durante La Colonización Intranasal Con Streptococcus pneumoniae

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing ...

Isolation, Purification and Labeling of Mouse Bone Marrow Neutrophils for Functional Studies and Adoptive Transfer Experiments

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or pathogenic effects depending on the inflammatory milieu. Nonetheless, despite the indispensable role of neutrophils in immunity, detailed understanding of the molecular factors that mediate neutrophils' effector and immunopathogenic effects in different infectious diseases and inflammatory conditions is still lacking, partly because of their short half life, the difficulties with handling of these cells and the lack of reliable experimental protocols for obtaining sufficient numbers of neutrophils for downstream functional studies and adoptive transfer experiments. Therefore, simple, fast, economical and reliable methods are highly desirable for harvesting sufficient numbers of mouse neutrophils for assessing functions such as phagocytosis, killing, cytokine production, degranulation and trafficking. To that end, we present a reproducible density gradient centrifugation-based protocol, which can be adapted in any laboratory to isolate large numbers of neutrophils from the bone marrow of mice with high purity and viability. Moreover, we present a simple protocol that uses CellTracker dyes to label the isolated neutrophils, which can then be adoptively transferred into recipient mice and tracked in several tissues for at least 4 hr post-transfer using flow cytometry. Using this approach, differential labeling of neutrophils from wild-type and gene-deficient mice with different CellTracker dyes can be successfully employed to perform competitive repopulation studies for evaluating the direct role of specific genes in trafficking of neutrophils from the blood into target tissues in vivo.

We describe a protocol for isolation and purification of neutrophils from mouse bone marrow by density gradient centrifugation and for neutrophil labeling using CellTracker dyes. This represents a simple, fast, reproducible and economical method for obtaining large numbers of neutrophils for downstream functional studies or adoptive transfer and tracking experiments.

Aislamiento, Purificación y Etiquetado de neutrófilos de médula ósea de ratón de Estudios funcionales y experimentos de transferencia adoptivos

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or ...

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of phenol soluble modulins (PSMs) and other toxins produced and secreted by Staphylococcus aureus on neutrophils. To study the effects of the PSMs on neutrophils we isolate fresh neutrophils using density gradient centrifugation. These neutrophils are loaded with a dye that fluoresces upon calcium mobilization. The activation of neutrophils by PSMs initiates a rapid and transient increase in the free intracellular calcium concentration. In a flow cytometry experiment this rapid mobilization can be measured by monitoring the fluorescence of a pre-loaded dye that reacts to the increased concentration of free Ca2+. Using this method we can determine the PSM concentration necessary to activate the neutrophil, and measure the effects of specific and general inhibitors of the neutrophil activation.	
	To investigate the expression of the PSMs in the intracellular space, we have constructed reporter fusions of the promoter of the PSM&alpha; operon to GFP. When these reporter strains of S. aureus are phagocytosed by neutrophils, the induction of expression can be observed using fluorescence microscopy.

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of PSMs and other toxins secreted by Staphylococcus aureus on neutrophils using flow cytometry and fluorescence microscopy.

El estudio de las interacciones de<em&gt; Staphylococcus aureus</em&gt; Con neutrófilos mediante citometría de flujo y microscopía Tiempo Lapso

We present methods to study the effect of phenol soluble modulins (PSMs)  and other toxins produced and secreted by Staphylococcus  aureus on neutrophils. ...

Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident immune cell populations in the oral mucosa and gingiva. We have established a technique for the isolation and study of immune cells from murine gingival tissues, an area of constant microbial exposure and a vulnerable site to a common inflammatory disease, periodontitis. Our protocol allows for a detailed phenotypic characterization of the immune cell populations resident in the gingiva, even at steady state. Our procedure also yields sufficient cells with high viability for use in functional studies, such as the assessment of cytokine secretion ex vivo. This combination of phenotypic and functional characterization of the gingival immune cell network should aid towards investigating the mechanisms involved in oral immunity and periodontal homeostasis, but will also advance our understanding of the mechanisms involved in local immunopathology.

We have established a technique for the isolation, phenotypic characterization and functional analysis of immune cells from murine gingiva.

El aislamiento, caracterización y examen funcional de la red celular gingival Inmune

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident ...

Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both microbial and dietary. Given an increasing understanding that these luminal antigens help shape the immune response and that education of immune cells within the intestine is critical for a number of systemic diseases, there has been increased interest in characterizing the intestinal immune system. However, many published protocols are arduous and time-consuming. We present here a simplified protocol for the isolation of lymphocytes from the small-intestinal lamina propria, intraepithelial layer, and Peyer&#39;s patches that is rapid, reproducible, and does not require laborious Percoll gradients. Although the protocol focuses on the small intestine, it is also suitable for analysis of the colon. Moreover, we highlight some aspects that may need additional optimization depending on the specific scientific question. This approach results in the isolation of large numbers of viable lymphocytes that can subsequently be used for flow cytometric analysis or alternate means of characterization.

There is growing interest in the quantitative characterization of intestinal lymphocytes owing to increasing recognition that these cells play a critical role in a variety of intestinal and systemic diseases. In this protocol, we describe how to isolate single cell populations from different small-intestinal compartments for subsequent flow cytometric characterization.

El aislamiento y la citometría de flujo Caracterización de murino Small intestinal linfocitos

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

Análisis de linfocitos extravasación El uso de una<em&gt; In Vitro</em&gt; Modelo de la barrera sangre-cerebro humano

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

Isolation and Characterization of Neutrophil-derived Microparticles for Functional Studies

Polymorphonuclear neutrophil-derived microparticles (PMN)-MPs) are lipid bilayer, spherical microvesicles with sizes ranging from 50&#8211;1,000 nm in diameter. MPs are a newly evolving, important part of cell-to-cell communication and signaling machinery. Because of their size and the nature of their release, until recently MP existence was overlooked. However, with improved technology and analytical methods their function in health and disease is now emerging. The protocols presented here are aimed at isolating and characterizing PMN-MPs by flow cytometry and immunoblotting. Moreover, several implementation examples are given. These protocols for MP isolation are fast, low-cost, and do not require the use of expensive kits. Furthermore, they allow for the labeling of MPs following isolation, as well as pre-labeling of source cells prior to MP release, using a membrane-specific fluorescent dye for visualization and analysis by flow cytometry. These methods, however, have several limitations including purity of PMNs and MPs and the need for sophisticated analytical instrumentation. A high-end flow cytometer is needed to reliably analyze MPs and minimize false positive reads due to noise or auto-fluorescence. The described protocols can be used to isolate and define MP biogenesis, and characterize their markers and variation in composition under different stimulating conditions. Size heterogeneity can be exploited to investigate whether the content of membrane particles versus exosomes is different, and whether they fulfill different roles in tissue homeostasis. Finally, following isolation and characterization of MPs, their function in cellular responses and various disease models (including, PMN-associated inflammatory disorders, such as Inflammatory Bowel Diseases or Acute Lung Injury) can be explored.

New protocols are described here to isolate and characterize microparticles derived from human and mouse neutrophils. These protocols utilize ultracentrifugation, flow cytometry, and immunoblotting techniques to analyze microparticle content, and they can be used to study the role of microparticles derived from various cell types in cellular function.

Aislamiento y caracterización de micropartículas de neutrófilos derivado de estudios funcionales

Polymorphonuclear neutrophil-derived microparticles (PMN)-MPs) are lipid bilayer, spherical microvesicles with sizes ranging from 50&#8211;1,000 nm in ...

Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays

Mast cells (MCs), as a part of the immune system, play a key role in defending the host against several pathogens and in the initiation of the allergic immune response. The activation of MCs via the cross-linking of surface IgE bound to high affinity IgE receptor (Fc&#949;RI), as well as through the stimulation of several other receptors, initiates the rise of the free intracellular Ca2+ level ([Ca2+]i) that promotes the release of inflammatory and allergic mediators. The identification of molecular constituents involved in these signaling pathways is crucial for understanding the regulation of MC function. In this article, we describe a protocol for the isolation of murine connective tissue type MCs by peritoneal lavage and cultivation of peritoneal MCs (PMCs). Cultures of MCs from various knockout mouse models by this methodology represent a useful approach to the identification of proteins involved in MC signaling pathways. In addition, we also describe a protocol for single cell Fura-2 imaging as an important technique for the quantification of Ca2+ signaling in MCs. Fluorescence-based monitoring of [Ca2+]i is a reliable and commonly used approach to study Ca2+ signaling events, including store-operated calcium entry, which is of utmost importance for MC activation. For the analysis of MC degranulation, we describe a &#946;-hexosaminidase release assay. The amount of &#946;-hexosaminidase released into the culture medium is considered as a degranulation marker for all three different secretory subsets described in MCs. &#946;-hexosaminidase can easily be quantified by its reaction with a colorigenic substrate in a microtiter plate colorimetric assay. This highly reproducible technique is cost-effective and requires no specialized equipment. Overall, the provided protocol demonstrates a high yield of MCs expressing typical MC surface markers, displaying typical morphological and phenotypic features of MCs, and demonstrating highly reproducible responses to secretagogues in Ca2+-imaging and degranulation assays.

Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays

Here, we present a protocol to isolate and cultivate murine peritoneal mast cells. We also describe two protocols for their functional characterization: a fluorescent imaging of intracellular free Ca2+ concentration and a degranulation assay based on colorimetric quantification of the released &#946;-hexosaminidase.

Aislamiento de mastocitos de peritoneo derivado y su caracterización funcional con Ca2 +-proyección de imagen y pruebas de degranulación

Mast cells (MCs), as a part of the immune system, play a key role in defending the host against several pathogens and in the initiation of the allergic ...