ZG is supported by grants from the I-CORE Program of The Israel Science Foundation (Grant No. 41/11), the Abisch-Frenkel Foundation, the Rosetrees Trust, the Israel Cancer Research Foundation (ICRF - Research Career Development Award) and the CONCERN foundation. ZGF is supported by grants from the Israel Cancer Research Foundation (ICRF - Research Career Development Award), Chief Scientist of the Israel Ministry of Health and the Israel Lung Association.

<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.

Neutrophils are the most abundant of all white blood cells and are the first responders in cases of infection and inflammation. As such, they are highly sensitive to external cues and are easily activated. In addition, neutrophils have a very short half-life and a rapid turnover. Together, these characteristics raise several difficulties in working with neutrophils, such that unique experimental strategies are required. For example, there are several neutrophil purification strategies, each with its own pros and cons.</p...

Neutrophils were initially characterized as the innate immune cells which serve as first line defense against invading microorganisms. Today it is known that neutrophils have more far-reaching functions, being involved in mounting adaptive immune responses against foreign antigens1,2, regulating hematopoiesis3, angiogenesis4 and wound healing5. In addition, neutrophils may affect tumor growth and metastatic progression by virtue of their pro- and anti-tumor activities6,7. Neutrophils are characterized by a polymorphic segmented nucleus (hence termed polymorphonuclear (PMN) leukocytes) and contain at least three distinct subclasses of granules as well as secretory vesicles8 (Figure 1A-C).
Neutrophils possess high phagocytic capacity and high NADPH oxidase activity critical for microbial elimination, and secrete a wide range of chemokines important for attraction of additional neutrophils and other immune cells to the site of inflammation8,9. Neutrophils are characterized by the expression of a large amount of surface receptors including Toll-like receptors (TLRs), C-type Lectin Receptors (CLRs), complement receptor 3 (CD11b/CD18) and other adhesion molecules (e.g., L-selectin, LFA-1, VLA-4 and carcinoembryonic antigen-related cell adhesion molecule 3 (CEACAM3/CD66b)), chemokine receptors (e.g., CXCR1, CXCR2, CCR1, CCR2), chemoattractant receptors (e.g., PAFR, LTB4R and C5aR), cytokine receptors (e.g., G-CSFR, IL-1R, IL-4R, IL-12R, IL-18R, TNFR), formyl-peptide receptors (e.g., FPR1-3), and Fc receptors (e.g., CD16 (Fc&#947;RIII), CD32 (Fc&#947;RII), and CD64 (Fc&#947;RI)10. In mice, neutrophils are usually identified as CD11b+Ly6G+, whereas human neutrophils are identified using the CD11b, CD15, CD16 and CD66b leukocyte markers. It is also generally accepted to stain for the granule proteins myeloperoxidase (MPO) and neutrophil elastase (NE) for detection of neutrophils in tissues.
It is still unclear whether the diverse functions of neutrophils are mediated by the same cell or by distinct cell sub-populations. Accumulating data suggest for the presence of a heterogenic neutrophil population that exhibits a high degree of plasticity affected by pro-inflammatory stimuli and the microenvironment11,12. Fridlender et al.13 have grossly divided the neutrophils in cancer into two major sub-populations termed N1 with anti-tumor properties and N2 with pro-tumor properties. In cancer, as well as in chronic inflammation, there is an additional sub-population composed of granulocytic myeloid-derived suppressor cells (G-MDSCs) that suppress T cell responses14. G-MDSCs are considered to be immature myeloid cells characterized by a CD11b+Ly6ClowLy6Ghi phenotype in mice15, while having a CD15+/CD16low phenotype in human16. G-MDSCs express higher levels of arginase and myeloperoxidase, while lower levels of cytokines and chemokines than normal circulating neutrophils. They are less phagocytic and migratory, but produce higher levels of ROS15,17,18. In the present paper we will describe some basic methodologies for isolation and characterization of neutrophils with anti-tumor properties.
While neutrophils constitute the largest population of all white blood cells in the human circulation (45 - 70%; 1,800 - 6,000/&#956;l), in mice, under normal conditions, they are rather sparse (10 - 15%; 300 - 500/&#956;l). The neutrophil count increases steadily upon inflammation and occasionally in cancer, which represents a state of chronic inflammation7. Neutrophils develop from multipotent common myeloid precursor (CMP) cells in the bone marrow, through a differentiation process passing the stages of myeloblasts (MB), promyelocytes (PM), myelocytes (MC), metamyelocytes (MM) and band cells (BC)8. The mature, post-mitotic neutrophils may remain within the bone marrow for 4 - 7 days before they are released to the circulation8. Neutrophil turnover in the blood is usually rapid with an average half-life of 6 - 12 hrs, which may be prolonged under inflammatory conditions. Unstimulated neutrophils have limited anti-tumorigenic activity, a feature that can be acquired by exposing the na&#239;ve neutrophils to the chemokines IL-8 (CXCL2), CCL2, CCL5 and CXCL56,19 or artificially, by exposing them to the phorbol ester phorbol 12-myristate 13-acetate (PMA)6.
The short half-life of blood neutrophils together with the low number of neutrophils (~ 3 - 5 x 105) achieved from 1 ml blood of a na&#239;ve 6 - 8 week old mouse, have made it difficult to explore the function of circulating mouse neutrophil in vitro. To overcome this difficulty, other sources have been used. For instance, large numbers of neutrophils may be obtained from the bone marrow20 or the peritoneum following the induction of sterile inflammation (e.g., after intraperitoneal injection of thioglycollate broth or Zymosan A). It should be noted that neutrophils obtained from the peritoneal cavity do not exert any anti-tumorigenic activity (unpublished observation).
Granot et al.6 observed that BALB/c mice inoculated orthotopically with the mouse 4T1 breast carcinoma cell line develop neutrophilia which aggravates with tumor progression6 (Figure 2A), such that 20 - 40 million blood neutrophils can be easily isolated from 1 ml blood 3 - 4 weeks post-tumor inoculation. These neutrophils have acquired anti-tumor activities, and have accordingly been coined tumor-entrained neutrophils (TEN), in order to distinguish them from na&#239;ve neutrophils6 (Figure 2B). While high-density neutrophils (HDN, Figure 1A) are highly anti-tumorigenic, low-density neutrophils (LDN, Figure 1B) generated in the context of cancer are not21. Also, high-density neutrophils from the bone marrow and spleen of tumor-bearing mice have anti-tumor activity (unpublished data). It should be noted that with tumor progression the spleen becomes gradually enlarged (splenomegaly), with increasing amounts of neutrophils.
It should be noted that TEN are also generated in other models of cancer including both spontaneous (MMTV-PyMT and MMTV-Wnt1 mammary tumors and k-Ras driven lung tumors) and injected (AT-3 (MMTV-PyMT) and E0771 breast carcinoma cells, LLC Lewis lung carcinoma cells and B16-F10 melanoma cells). However, the extent of neutrophil mobilization in these tumor models is far less than of 4T1-inoculated mice, reaching 5 - 10 x 106 neutrophils in 1 ml blood after 3 weeks.

<table><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>PLASTIC WARES AND EQUIPMENTS</td><td></td><td></td><td></td></tr><tr><td>24-well Tissue Culture Plate&amp;nbsp;</td><td>Falcon</td><td>353047</td><td>Sterile</td></tr><tr><td>100 mm Tissue Culture Plate&amp;nbsp;</td><td>Corning</td><td>430167</td><td>Sterile</td></tr><tr><td>25 cm&lt;sup&gt;2&lt;/sup&gt; Tissue Culture Flask</td><td>Nunc</td><td>156340</td><td>Sterile</td></tr><tr><td>90 mm Bacterial Grade Culture Dish&amp;nbsp;</td><td>Miniplast, Ein Shemer, Israel</td><td>20090-01-017</td><td>Sterile</td></tr><tr><td>15 ml Sterile Conical Centrifuge Tube&amp;nbsp;</td><td>Miniplast, Ein Shemer, Israel</td><td>835015-40-111</td><td>Sterile</td></tr><tr><td>50 ml Sterile Conical Centrifuge Tube&amp;nbsp;</td><td>Miniplast, Ein Shemer, Israel</td><td>835050-21-111</td><td>Sterile</td></tr><tr><td>Falcon 12 x 75 mm Round-Bottom Polystyrene Tube&amp;nbsp;</td><td>Becton Dickinson</td><td>352058</td><td>Sterile</td></tr><tr><td>Millicell 24 Migration Plate with a pore size of 5&amp;mu;m&amp;nbsp;</td><td>Merck Millipore</td><td>PSMT010R1</td><td>Sterile</td></tr><tr><td>White 96-Flat-Bottom Well Plate&amp;nbsp;</td><td>Costar</td><td>3917</td><td>Sterile</td></tr><tr><td>Cell Strainer (40 mm)&amp;nbsp;</td><td>BD Falcon</td><td>352340</td><td>Sterile</td></tr><tr><td>20G x 1.5&quot; Needle</td><td>BD Microlance 3&amp;nbsp;</td><td>301300</td><td>Sterile</td></tr><tr><td>23G x 1&quot; Needle&amp;nbsp;</td><td>BD Microlance 4</td><td>300800</td><td>Sterile</td></tr><tr><td>25G x 5/8&quot; Needle&amp;nbsp;</td><td>BD Microlance 5</td><td>300600</td><td>Sterile</td></tr><tr><td>0.3 ml Syringe with a 30G x 8mm Needle</td><td>BD Micro-Fine Plus Demi</td><td>320829</td><td>Sterile</td></tr><tr><td>9 mm Clips&amp;nbsp;</td><td>BD, AutoClip&amp;nbsp;</td><td>427631</td><td>Sterile</td></tr><tr><td>EasySep Magnet&amp;nbsp;</td><td>STEMCELL Technologies</td><td>18000</td><td></td></tr><tr><td>MACS LS Separation Column&amp;nbsp;</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&amp;nbsp;</td><td>Menzel-Gl&amp;auml;ser Superfrost&amp;nbsp; Plus Thermo</td><td>J1800AMNZ</td><td></td></tr><tr><td>Orbital Shaker&amp;nbsp;</td><td>Sky line, ELMI</td><td>S-3.02.10L</td><td></td></tr><tr><td>Plate Reader&amp;nbsp;</td><td>TECAN</td><td>InfiniteF200Pro</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)&amp;nbsp;</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&amp;nbsp;</td><td>Sigma</td><td>H3149</td><td></td></tr><tr><td>Sodium azide (NaN&lt;sub&gt;3&lt;/sub&gt;)</td><td>Sigma</td><td>S8032</td><td>Highly toxic, handle with care</td></tr><tr><td>Thioglycollate powder&amp;nbsp;</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>MEDIA AND SUPPLEMENTS</td><td></td><td></td><td></td></tr><tr><td>Dulbecco&#039;s modified Eagle medium (DMEM)</td><td>Sigma</td><td>D5796</td><td>Sterile</td></tr><tr><td>Opti-MEM&amp;reg; I reduced serum medium&amp;nbsp;</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 x 1000 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 Mg&lt;sup&gt;2+&lt;/sup&gt; and Ca&lt;sup&gt;2+&lt;/sup&gt;&amp;nbsp;</td><td>Biological Industries, Beth HaEmek, Israel</td><td>02-023-1</td><td>Sterile</td></tr><tr><td>PBS x 10 without Ca&lt;sup&gt;2+&lt;/sup&gt; and Mg&lt;sup&gt;2+&lt;/sup&gt;&amp;nbsp;</td><td>Biological Industries, Beth HaEmek, Israel</td><td>02-023-5A</td><td>Sterile</td></tr><tr><td>HPLC grade water&amp;nbsp;</td><td>J.T. Baker</td><td>4218-03</td><td>Autoclave</td></tr><tr><td>SOLUTIONS</td><td></td><td></td><td></td></tr><tr><td>ACK &amp;ndash; Ammonium-Chloride-Potassium</td><td>Life Technologies&amp;nbsp;</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 -20&lt;sup&gt;o&lt;/sup&gt;C.</td></tr><tr><td>Eosin Y solution</td><td>Sigma</td><td>HT110-2-32</td><td></td></tr><tr><td>Hanks&#039; 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.&amp;nbsp;</td></tr><tr><td>Histopaque-1119&amp;nbsp;</td><td>Sigma</td><td>11191</td><td>Sterile filter through a 0.2 mm filter.</td></tr><tr><td>Histopaque-1077&amp;nbsp;</td><td>Sigma</td><td>10771</td><td>Sterile filter through a 0.2 mm filter.</td></tr><tr><td>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&#039;s Hematoxylin solution&amp;nbsp;</td><td>Sigma</td><td>MHS-32</td><td></td></tr><tr><td>PBS + 0.5% BSA</td><td></td><td></td><td>Dissolve 2.5 g 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>Dissolve 12.5g BSA in a mixture of 250 ml sterile HPLC-grade water 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>Dissolve 250 mg BSA in 50 ml sterile PBS 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% NaN&lt;sub&gt;3&lt;/sub&gt;)</td><td></td><td></td><td>Dissolve 250 mg BSA in 50 ml sterile PBS&amp;nbsp; and add 200 ml of 0.5M EDTA pH 8.0 and 500 ml of 2% NaN&lt;sub&gt;3&lt;/sub&gt;, 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 1,000 ml ddw, autoclave</td></tr><tr><td>0.2% NaCl solution</td><td></td><td></td><td>Dissolve 2 g NaCl in 1,000 ml ddw, autoclave</td></tr><tr><td>1.6% NaCl solution</td><td></td><td></td><td>Dissolve 16 g NaCl in 1,000 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 4&lt;sup&gt;o&lt;/sup&gt;C. Highly toxic.</td></tr><tr><td>3% Thioglycollate solution</td><td></td><td></td><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Dissolve 3 g of thioglycollate powder in 100 ml ddw.&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 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&amp;nbsp;</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>Resuspend 1 mg Zymosan A in 1 ml sterile PBS in an Eppendorf tube. Vortex vigorously and incubate the tube at 37 &lt;sup&gt;o&lt;/sup&gt;C for 30 min. Do not autoclave. Prepare the solution freshly before use.</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&amp;nbsp;</td><td>STEMCELL Technologies</td><td>18151</td><td></td></tr><tr><td>the EasySep magnetic nanoparticles&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</td><td>Cayman Chemical Company&amp;nbsp;</td><td>500290</td><td></td></tr><tr><td>ANTIBODIES</td><td></td><td></td><td></td></tr><tr><td>FcR blocking antibody&amp;nbsp;</td><td>Biolegend</td><td>101302</td><td></td></tr><tr><td>Purified rat anti-Ly6G antibody&amp;nbsp;</td><td>BD Pharmingen&amp;nbsp;</td><td>551459</td><td>Clone 1A8</td></tr><tr><td>PE-conjugated rat anti-mouse Ly6G antibody&amp;nbsp;</td><td>Biolegend</td><td>127608</td><td>Clone 1A8</td></tr><tr><td>FITC-conjugated rat anti-mouse Ly6G</td><td>BD Pharmingen&amp;nbsp;</td><td>551460</td><td>Clone 1A8</td></tr><tr><td>PerCP-Cy5.5 rat anti-mouse Ly6G&amp;nbsp;</td><td>TONBO Biosciences</td><td>65-1276</td><td>Clone 1A8</td></tr><tr><td>violetFluor 450-conjugated rat anti-mouse Ly6G&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</td><td>553127</td><td>Clone RB6-8C5</td></tr><tr><td>PE-conjugated rat anti-mouse CD45</td><td>BD Pharmingen&amp;nbsp;</td><td>553081</td><td>Clone 30-F11</td></tr><tr><td>FITC-conjugated rat anti-mouse F4/80&amp;nbsp;</td><td>Abcam</td><td>ab60343</td><td>Clone BM8</td></tr><tr><td>FITC-conjugated mouse anti-human CD66b&amp;nbsp;</td><td>Biolegend</td><td>305103</td><td>Clone G10F5</td></tr><tr><td>Purified rat isotype control antibody (IgG&lt;sub&gt;2a&lt;/sub&gt;, k)&amp;nbsp;</td><td>BD Pharmingen&amp;nbsp;</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

Neutrophils, the most abundant of all white blood cells in the human circulation, play an important role in the host defense against invading microorganisms. In addition, neutrophils play a central role in the immune surveillance of tumor cells. They have the ability to recognize tumor cells and induce tumor cell death either through a cell contact-dependent mechanism involving hydrogen peroxide or through antibody-dependent cell-mediated cytotoxicity (ADCC). Neutrophils with anti-tumor activity can be isolated from peripheral blood of cancer patients and of tumor-bearing mice. These neutrophils are termed tumor-entrained neutrophils (TEN) to distinguish them from neutrophils of healthy subjects or na&#239;ve mice that show no significant tumor cytotoxic activity. Compared with other white blood cells, neutrophils show different buoyancy making it feasible to obtain a &#62; 98% pure neutrophil population when subjected to a density gradient. However, in addition to the normal high-density neutrophil population (HDN), in cancer patients, in tumor-bearing mice, as well as under chronic inflammatory conditions, distinct low-density neutrophil populations (LDN) appear in the circulation. LDN co-purify with the mononuclear fraction and can be separated from mononuclear cells using either positive or negative selection strategies. Once the purity of the isolated neutrophils is determined by flow cytometry, they can be used for in vitro and in vivo functional assays. We describe techniques for monitoring the anti-tumor activity of neutrophils, their ability to migrate and to produce reactive oxygen species, as well as monitoring their phagocytic capacity ex vivo. We further describe techniques to label the neutrophils for in vivo tracking, and to determine their anti-metastatic capacity in vivo. All these techniques are essential for understanding how to obtain and characterize neutrophils with anti-tumor function.

In a recent study we identified an anti-metastatic function for neutrophils6. Neutrophils from tumor-bearing mice acquire a cytotoxic phenotype and have the capacity to kill tumor cells6. This is in contrast to neutrophils from na&#239;ve mice that have no significant anti-tumor effect6. Several of the techniques described in the Protocol Section have been used for studying anti-tumor neutrophil function in vitro and in vivo6.
Tumor...

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.

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

24.7K Views.  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.

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 ...

Neutrophil Extracellular Traps Generated by Low Density Neutrophils Obtained from Peritoneal Lavage Fluid Mediate Tumor Cell Growth and Attachment

Activated neutrophils release neutrophil extracellular traps (NETs), which can capture and destroy microbes. Recent studies suggest that NETs are involved in various disease processes, such as autoimmune disease, thrombosis, and tumor metastases. Here, we show a detailed in vitro technique to detect NET activity during the trapping of free tumor cells, which grow after attachment to NETs. First, we collected low density neutrophils (LDN) from postoperative peritoneal lavage fluid from patients who underwent laparotomies. Short-term culturing of LDN resulted in massive NET formation that was visualized with green fluorescent nuclear and chromosome counterstain. After co-incubation of human gastric cancer cell lines MKN45, OCUM-1, and NUGC-4 with the NETs, many tumor cells were trapped by the NETs. Subsequently, the attachment was completely abrogated by the degradation of NETs with DNase I. Time-lapse video revealed that tumor cells trapped by the NETs did not die but instead grew vigorously in a continuous culture. These methods may be applied to the detection of adhesive interactions between NETs and various types of cells and materials.

Here, we present a method in which human low-density neutrophils (LDN), recovered from postoperative peritoneal lavage fluid, produce massive neutrophil extracellular traps (NETs) and efficiently trap free tumor cells that subsequently grow.

Activated neutrophils release neutrophil extracellular traps (NETs), which can capture and destroy microbes. Recent studies suggest that NETs are involved ...

Cancer Research

Transfer of Manipulated Tumor-associated Neutrophils into Tumor-Bearing Mice to Study their Angiogenic Potential In Vivo

The contribution of neutrophils to the regulation of tumorigenesis is getting increased attention. These cells are heterogeneous, and depending on the tumor milieu can possess pro- or anti-tumor capacity. One of the important cytokines regulating neutrophil functions in a tumor context are type I interferons. In the presence of interferons, neutrophils gain anti-tumor properties, including cytotoxicity or stimulation of the immune system. Conversely, the absence of an interferon signaling results in prominent pro-tumor activity, characterized with strong stimulation of tumor angiogenesis. Recently, we could demonstrate that pro-angiogenic properties of neutrophils depend on the activation of nicotinamide phosphoribosyltransferase (NAMPT) signaling pathway in these cells. Inhibition of this pathway in tumor-associated neutrophils leads to their potent anti-angiogenic phenotype. Here, we demonstrate our newly established model allowing in vivo evaluation of tumorigenic potential of manipulated tumor-associated neutrophils (TANs). Shortly, pro-angiogenic tumor-associated neutrophils can be isolated from tumor-bearing interferon-deficient mice and repolarized into anti-angiogenic phenotype by blocking of NAMPT signaling. The angiogenic activity of these cells can be subsequently evaluated using an aortic ring assay. Anti-angiogenic TANs can be transferred into tumor-bearing wild type recipients and tumor growth should be monitored for 14 days. At day 14 mice are sacrificed, tumors removed and cut with their vascularization assessed. Overall, our protocol provides a novel tool to in vivo evaluate angiogenic capacity of primary cells, such as tumor-associated neutrophils, without a need to use artificial neutrophil cell line models. vc

Here, we show therapeutic potential of anti-angiogenic tumor-associated neutrophils after their transfer into tumor-bearing mice. This protocol can be used to manipulate neutrophil activity ex vivo and to subsequently evaluate their functionality in vivo&#160;in developing tumors. It is an appropriate model for studying potential neutrophil-based immunotherapies.

The contribution of neutrophils to the regulation of tumorigenesis is getting increased attention. These cells are heterogeneous, and depending on the ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

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.

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

Isolation of Immune Cells from Primary Tumors

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various chemokines and growth factors 1,2. However, once in the TME, these cells lose the ability to promote anti-tumor immunity and begin to support tumor growth and down-regulate anti-tumor immune responses 3-4. Studies on tumor-associated leukocytes have mainly focused on cells isolated from tumor-draining lymph nodes or spleen due to the inherent difficulties in obtaining sufficient cell numbers and purity from the primary tumor. While identifying the mechanisms of cell activation and trafficking through the lymphatic system of tumor bearing mice is important and may give insight to the kinetics of immune responses to cancer, in our experience, many leukocytes, including dendritic cells (DCs), in tumor-draining lymph nodes have a different phenotype than those that infiltrate tumors 5,6 . Furthermore, we have previously demonstrated that adoptively-transferred T cells isolated from the tumor-draining lymph nodes are not tolerized and are capable of responding to secondary stimulation in vitro unlike T cells isolated from the TME, which are tolerized and incapable of proliferation or cytokine production 7,8. Interestingly, we have shown that changing the tumor microenvironment, such as providing CD4+ T helper cells via adoptive transfer, promotes CD8+ T cells to maintain pro-inflammatory effector functions 5. The results from each of the previously mentioned studies demonstrate the importance of measuring cellular responses from TME-infiltrating immune cells as opposed to cells that remain in the periphery. To study the function of immune cells which infiltrate tumors using the Miltenyi Biotech isolation system9, we have modified and optimized this antibody-based isolation procedure to obtain highly enriched populations of antigen presenting cells and tumor antigen-specific cytotoxic T lymphocytes. The protocol includes a detailed dissection of murine prostate tissue from a spontaneous prostate tumor model (TRansgenic Adenocarcinoma of the Mouse Prostate -TRAMP) 10 and a subcutaneous melanoma (B16) tumor model followed by subsequent purification of various leukocyte populations.

In this report, we describe a protocol for isolating highly purified populations of leukocytes that infiltrate tumors. This protocol is adapted from the Miltenyi Biotech protocol to enhance yield and purity for isolating cells from complex tumor tissue.

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various ...

Biology

Isolation of Human Neutrophils from Whole Blood and Buffy Coats

Neutrophils (PMNs) are the most abundant leukocytes in human circulation, ranging from 40 to 70% of total blood leukocytes. They are the first cells recruited at the site of inflammation via rapid extravasation through vessels. There, neutrophils perform an array of functions to kill invading pathogens and mediate immune signaling. Freshly purified neutrophils from human blood are the model of choice for study, as no cell line fully replicates PMN functions and biology. However, neutrophils are short-lived, terminally differentiated cells and are highly susceptible to activation in response to physical (temperature, centrifugation speed) and biological (endotoxin, chemo- and cytokines) stimuli. Therefore, it is crucial to follow a standardized, reliable, and fast method to obtain pure and non-activated cells. This protocol presents an updated protocol combining density gradient centrifugation, red blood cell (RBC) sedimentation, and RBC lysis to obtain high PMN purity and minimize cell activation. Furthermore, methods to assess neutrophil isolation quality, viability, and purity are also discussed.

This protocol details a method for the isolation of neutrophils from whole blood, buffy coats, or leukapheresis membranes, achieving good yield, high purity, and minimal cell activation. We utilize gradient purification, red blood cell (RBC) sedimentation, and RBC lysis to obtain a high-quality/purity neutrophil preparation.

Neutrophils (PMNs) are the most abundant leukocytes in human circulation, ranging from 40 to 70% of total blood leukocytes. They are the first cells ...

Isolation of Cytotoxic Neutrophils from Tumor-Bearing Mouse Blood

Source: Sionov, R. V., et al., <a href="https://app.jove.com/v/52933">Isolation and Characterization of Neutrophils with Anti-Tumor Properties.</a> J. Vis. Exp. (2015)
In this video, we demonstrate the isolation of cytotoxic high-density neutrophils and low-density neutrophils from the blood of a tumor-bearing mouse by discontinuous sucrose gradient centrifugation.

Source: Sionov, R. V., et al., Isolation and Characterization of Neutrophils with Anti-Tumor Properties. J. Vis. Exp. (2015)
In this video, we demonstrate ...

JoVE Encyclopedia of Experiments

Immunology

Immune Systems and Components

Cell Isolation and Culture

Determining the Role of Neutrophil-Like Cells in Lymphoma Cell Sensitivity to a Test Drug

Source: Hirz, T., et al. <a href="https://app.jove.com/v/53846">Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents.</a> J. Vis. Exp. (2016).
This video demonstrates an assay investigating the role of neutrophil-like cells in safeguarding lymphoma cells against the cytotoxicity of chemotherapeutic agents. A 3D co-culture, consisting of lymphoma and neutrophil-like cells, is established and exposed to vincristine&#8212;a chemotherapy drug. Direct contact with the neutrophil-like cells induces an upregulation of anti-apoptotic proteins in the lymphoma cells, thereby promoting their survival in the presence of vincristine.

1. Differentiation of HL60 Cells along the Granulocytic Pathway and Their Coculture with RL Lymphoma B Cells in 3D Model

	Differentiation of Human ...

Immunodiagnostics

Cellular Immunodiagnostics

Generating Anti-Angiogenic Tumor-Associated Neutrophils

For tumor-associated neutrophil isolation, place five tumors per well into individual wells of a sterile six-well plate, as they are harvested, and use sterile scissors to mince the tumors into 2- to 3-millimeter pieces. Next, digest the tumor fragments with 1 milliliter of disk-based collagenase, DNase I solution for 45 minutes at 37 degrees Celsius and 5% carbon dioxide with humidity, mixing the samples with a 10-milliliter syringe every 15 minutes.
At the end of the incubation, filter the cells through 100-micrometer strainers into one 15-milliliter tube per well to remove the undigested fibers, and add PBS to a final volume of 15 milliliters. Collect the dissociated cells by centrifugation, and lyse the red blood cells with 1 milliliter of lysis buffer per tube.
After mixing, combine the contents from each tube into a single 15-milliliter tube, stopping the reaction after two minutes with 11 milliliters of complete 4 degrees Celsius medium. After centrifugation, resuspend the pellet in 1 milliliter of PBBS, and block any nonspecific binding with three microliters of FC-blocked antibody for 15 minutes on ice.
At the end of the incubation, label the cells with anti-CD11b and Ly6G antibodies and any other antibodies of interest plus DAPI for a 30-minute incubation on ice protected from light. At the end of the incubation, wash the cells in 14 milliliters of PBS, and resuspend the pellets at a 1 x 107 cells per milliliter of fresh complete medium concentration on ice.
Then, sort the CD11b-positive Ly6G-high DAPI-negative neutrophils on a fluorescence-activated cell sorter according to standard sorting protocols, checking the purity of the isolated neutrophils on the flow cytometer at the end of the sort. For in vitro tumor-associated, neutrophil inhibition, seed 1.5 times 10 to the fifth sorted neutrophils into each of two wells of a 96-well U-bottom plate, and add FK866 to a final concentration of 100 nanomolar in complete medium into one well and an equal volume of complete medium alone to the other well. After two hours in the cell culture incubator, wash the cells two times with PBS, and resuspend the pellets in PBS at the appropriate concentration for the subsequent injection.