Name: development and identification of a novel subpopulation of human neutrophil-derived giant phagocytes in vitro
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The authors thank Dr. Edith Suss-Toby for her invaluable help with the confocal microscopy studies. This study was supported by the Ministry of Immigration Absorption and the Committee for Planning and Budgeting of the Council for Higher Education under the framework of the KAMEA program (LD and AP). We also gratefully acknowledge the support of the Research Fellow from the Lady Davis Foundation Post-Doctoral Research Fellowship (OR).

The protocol was approved by the local Human Rights Committee according to the declaration of Helsinki, and all participants signed an informed consent form.
1. Neutrophil Isolation and Development of G&#981; in Culture
NOTE: All steps should be performed using sterile tissue grade lipopolysaccaride (LPS)-free solutions in a Bio-Safety Laminar flow hood. Do not add antibiotics, cytokines&#160;or growth factors to the Roswell park memorial institute (RPMI)-1640 medium.
<ol>
	<li>Obtain at least 40 ml venous blood from young healthy adults using a sterile scalp vein set. Draw blood into vacutainer tubes containing ethylenediamine tetra acetic acid K3 salt (K3EDTA) and mix gently. Keep the blood at room temperature.</li>
	<li>Isolate the neutrophils by two step discontinuous density gradient using polysucrose at 1.119 and 1.077 g/ml. Bring solutions to room temperature before using. 
	NOTE: During centrifugation, red blood cells (RBCs) are aggregated by the polysucrose and sediment rapidly. The mononuclear cells (monocytes/lymphocytes) are found between the upper plasma/polysucrose -1,077 interface, whereas the neutrophils are found just above the RBCs, at the polysucrose -1,077/1,119 interface (see Figure 2). This method allows simultaneous separation of mononuclear cells and neutrophils from the same individual.</li>
</ol>
<img alt="Figure 2" src="/files/ftp_upload/54826/54826fig2.jpg" /> 
Figure 2: Neutrophil Isolation from Human Whole Blood. Polysucrose at a 1.077 g/ml is carefully layered on top of polysucrose-1.119 g/ml to form a discontinuous gradient. The diluted whole blood is then layered on top of the polysucrose-1.077. The tubes are immediately subjected to centrifugation at 700 x g for 30 min, at room temperature without brake. Three distinct bands are noted. (A) Mononuclear cells, (B) polymorphonuclear cells (PMN), and (C) red blood cells (RBC) at the bottom of the tube. <a href="https://cloudfront-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54826/54826fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol style="margin-left: 40px;">
	<li>Add 12 ml polysucrose-1119 to the bottom of a 50 ml sterile polypropylene conical centrifuge tube.</li>
	<li>Carefully layer 12 ml of polysucrose-1077 onto the polysucrose -1119.</li>
	<li>Dilute 10 - 12 ml whole blood to a final volume of 24 ml blood with ion free phosphate buffered saline (PBS) containing 2 % heat inactivated fetal calf serum (HI-FCS). Carefully layer 24 ml of the diluted whole blood onto the upper gradient of the tube.</li>
	<li>Centrifuge at 700 x g for 30 min at room temperature (20 - 24 &#176;C) without brake. 
	NOTE: Centrifugation at lower temperatures may result in cell clumping and poor recovery.</li>
	<li>Carefully remove the tubes from the centrifuge without disturbing the gradient. Two opaque layers should be observed (A: Mononuclear cells and B: PMN, depicted in Figure 2).</li>
	<li>Aspirate and discard the fluid up to 0.5 cm above layer A. Transfer (or discard) the cells from this layer to a tube marked &#34;Mononuclear&#34;.</li>
	<li>Aspirate and discard the remaining fluid up to 0.5 cm above layer B. Transfer the cells from this layer to a tube labeled &#34;PMN&#34;.</li>
	<li>Pool PMN from each two gradient tubes and wash with PBS containing 2% HI-FCS to a final volume of 30 ml. Centrifuge for 12 min at 200 x g, remove the supernatant and discard.</li>
	<li>To get rid of contaminating red blood cells (RBC), add 3 ml of hypotonic 0.2% ice cold sterile NaCl while resuspending the pellet by gently drawing in and out with a 1 ml sterile pipet tip. Keep on ice for 30 sec.</li>
	<li>After 30 s, restore isotonicity by adding 3 ml of sterile 1.6% ice cold NaCl to the tube.</li>
	<li>To the 6 ml of isotonic saline, add 6 ml of pre-warmed (37 &#176;C) RPMI-1640 medium supplemented with 2% HI-FCS and centrifuge at 250 x g for 12 min. Discard the supernatant. The PMN pellet should be clean of RBC contamination. 
	NOTE: If contaminated by RBC, the PMN pellet appears reddish.</li>
	<li>If some contaminating RBC remain, repeat steps 9 and 10 once more.</li>
	<li>Resuspend the cell pellet in 4 ml RPMI-1640 supplemented with 10% HI-FCS and count the cells to determine their concentration and viability by trypan blue exclusion.</li>
	<li>Adjust the concentration to 1.25 - 1.5 x 106 PMN/ml (depending on the experimental needs), and plate 1.0 mL/well in a 24 well plate. 
	NOTE: The purity of neutrophils in the granulocyte population always exceeded 95%, as assessed by May Grunewald-Giemsa staining and light microscopy.</li>
	<li>After seeding, place the cells in a humidified 5% CO2 incubator at 37 &#176;C.</li>
	<li>Replace medium every 3 days by gently aspirating half of the medium and adding the same volume of fresh RPMI-1640 medium supplemented with 10% HI-FCS. Use LPS free solutions and compounds and low LPS levels in HI-FCS (0.05 ng/ml or less). 
	NOTE: A gentle medium change is imperative since the G&#981;, which develop in culture do not firmly attach to the culture dish and vigorous washing may also wash out the developing cells. The appearance of G&#981; is noticeable at 3 - 4 days after PMN culturing, depending on the blood donor. Most of the analyses and assays described here are performed between 6 - 7 days in culture, when G&#981; are very large in size. It should be noted that addition of 1 - 10 ng/ml LPS to the RPMI-1640 medium did not affect G&#981; development in culture.14</li>
</ol>
2. Confocal Laser Scanning Microscopy
<ol style="margin-left: 40px;">
	<li>Prepare cytospins16 from freshly isolated neutrophils, and from the 7 day developed G&#981; cultures (prepared in section 1). 
	NOTE: To increase the concentration of G&#981; in the dish for various analyses, gently remove half of the medium. Make sure that G&#981; are not detected in the removed medium by examining the medium under a light microscope. Then, intensively pipet the remaining medium to remove lightly adhered G&#981;. Centrifuge the medium for 10 min at 200 x g, and resuspend the pellet in 100 - 120 &#181;l medium.
	<ol>
		<li>Use 100 - 120 &#181;l of the medium containing cells for each slide. Prepare duplicate or triplicate slides from each treatment. Spin for 7 min at 84 x g.</li>
	</ol>
	</li>
	<li>Dry the spun cells and fix the cells with 4 % paraformaldehyde at room temperature for 10 min under a chemical hood. Wash 3x with PBS (~100 &#181;l for a few seconds per wash). For intracellular staining, permeabilize cells with 0.5% Triton X-100 in PBS at room temperature for 10 min and wash 5x with PBS. 
	NOTE: At all stages, use appropriate buffer/solution volume to cover the perimeter of the cells on the slide. Use a hydrophobic barrier pen for determining cells perimeter. 
	Caution: Paraformaldehyde is toxic. Avoid contact with skin and eyes. Wear appropriate personal protective equipment.</li>
	<li>Block cells with 10% normal goat serum in RPMI-1640 medium and incubate overnight at 4 &#186;C or at room temperature for 40 min. Wash with PBS.</li>
	<li>Incubate with single antibody (Ab) or a combination of mouse and rabbit primary antibodies (Abs) at a 1:100 dilution (~100 &#181;l). Incubate overnight (18 - 20 hr) at 4 &#176;C. 
	NOTE: Here, mouse monoclonal Abs included: anti-CD14, anti-CD63, anti-CD66b, anti-CD1c, anti- CD15, and anti-Cytochrome b-245 light chain (p22-phox identification). Rabbit polyclonal Abs included: anti-CD68, anti-CD36, anti-LC3B, anti-Myeloperoxidase, anti-neutrophil elastase (NE), and anti-Nox2/gp91-phox Abs. Isotype controls included purified mouse IgG1 and IgG2, and rabbit IgG. Prepare the Abs according to manufacturer&#39;s instructions and use appropriate volume (about 100 &#181;l) to cover the perimeter of the cells.</li>
	<li>Wash the cells and incubate with 1/400 secondary antibodies Cy2-CF (488A)-conjugated goat anti-rabbit IgG (green) and/or Cy5 (CF 647)-conjugated goat anti-mouse IgG (red) at room temperature for 40 min. 
	NOTE: Dilute and prepare Abs according to manufacturer&#39;s instructions.</li>
	<li>After washing, mount slides with one drop of mounting medium, containing 4&#39;,6-diamidino-2-phenylindole (DAPI) for nuclear staining, then immediately place the cover slip.</li>
	<li>Analyze the slides by a confocal laser scanning system using fluorescence microscope and Plan Apo 40X immersion oil objective. Perform the analysis within 30 min to 2 h after preparation of the slides or keep at 4 &#176;C overnight.
	<ol>
		<li>Calculate the cell area and fluorescence intensity using an imaging software (e.g. Image J). For co-localization, quantify by software using Manders Overlap Coefficient (MOC)17. 
		NOTE: Only cells with MOC &#62;0.6 can be considered as cells with significant co-localization.</li>
	</ol>
	</li>
</ol>
3. Transmigration of PMN Across Endothelial Cells: Effects of IL-8 on Giant Phagocyte (G&#981;) Formation
NOTE: Use 24-well permeable cell culture inserts for the cell transmigration assay.
<ol style="margin-left: 40px;">
	<li>Coat the upper chamber of the insert with 150 &#181;l fibronectin at a concentration of 50 &#181;g/ml, and keep at room temperature for 30 min.</li>
	<li>Add to the upper chamber 5 x 104 EA.hy926 endothelial cells/well, resuspended in 150 &#181;l of formulated Dulbecco&#39;s Modified Eagle&#39;s Medium (complete growth medium). 
	NOTE: Ensure that the endothelial monolayer is confluent before use.</li>
	<li>To the lower chamber, add 700 &#181;L of the complete growth medium.</li>
	<li>Place the permeable cell culture inserts in cluster trays and culture the EA.hy926 endothelial cells for 2 days at 37 &#176;C in 5% CO2. 
	NOTE: In parallel, on the second day prepare fresh PMN (as described in section 1).</li>
	<li>After 2 days, replace the medium in the lower and upper chambers of the inserts.
	<ol>
		<li>To the lower chamber, add RPMI-1640 medium supplemented with 10% IH-FCS and interleukin (IL)-8 at a final concentration of 50 nM/ml. Do not add IL-8 to control lower chambers.</li>
		<li>To each upper chamber, add 106 fresh PMN in 100 &#181;l of RPMI-1640 medium supplemented with 10% IH-FCS.</li>
	</ol>
	</li>
	<li>Incubate the cluster trays at 37 &#176;C in 5% CO2 for 90 min.</li>
	<li>After 90 min incubation, remove the cells from the upper and the lower chambers separately and count each subpopulation. Express cells in each chamber as a percentage of the total cells added. 
	NOTE: Carefully remove cells from the upper chamber by pipetting gently in order to avoid removing endothelial cells and transfer to a sterile tube. Remove the cells from the lower chamber by pipetting and washing the lower chamber with 500 &#181;l and transfer to a second sterile tube.</li>
	<li>Pool 106 cells from several wells of transmigrating (lower chamber) and non-migrating (upper chamber) PMN fractions and culture each for 7 days without growth factors as specified in steps 14 - 16 (section 1).</li>
	<li>Spin cells onto slides16 and analyze the developed cells in each culture condition by confocal microscopy as described in section 2.</li>
</ol>

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The authors have nothing to disclose.

Giant phagocytes (G&#981;) are a newly defined subpopulation of neutrophil-derived cells expressing fundamental and specific neutrophilic markers such as CD66b/CD15/CD63/MPO/NE. This type of neutrophil-derived phagocyte was not described in the literature before. Unlike neutrophils that are short-lived and undergo apoptosis, G&#981; are Annexin-V-negative and display extended lifespan. Yet, like neutrophils, G&#981; also internalize particles and produce NADPH oxidase-dependent ROS in response to those particles and to PMA. However, their abilities to internalize OxLDL and consequently to produce ROS are unique features of G&#981;.14
A number of factors were shown to influence their development in culture. The lack of external cytokines or growth factors in the growth medium is essential (specifically GM-CSF/IL-4). However, neutrophils migration towards IL-8 proved a discriminating factor between those that developed into G&#981; and those that did not. Also, internalization of debris arising from apoptotic neutrophils, the expression of autophagy proteins (LC3B) and functional NADPH oxidase, were all shown to be imperative for their development, since their inhibition prevented G&#981; formation (Figure 1). Apparently, the development of these giant cells arising from neutrophils differs from that characterizing giant cell formation in the monocyte/macrophage lineage. The latter form multi-nucleated giant cells associated with diverse chronic inflammatory diseases,20,21 whereas the neutrophilic G&#981; described here develop via autophagocytosis, by engulfing cell remnants and remain mostly mono-nucleated throughout their development,14 (rarely however, sometimes a second nucleus can be observed). Moreover, a number of controls established their neutrophilic origin: (1) expression of the specific neutropilic markers and absence of dendritic and monocytic lineage markers, (2) their hampered development in monocytes/PMN co-cultures, (3) their different patterns of movement in culture from macrophages (as evidenced by live cell imaging and time-lapse microscopy),14 (4) their light adherence to plastic dishes and (5) their development from pure CD15+/CD14- PMN acquired by flow cytometry.
Some of the functions identified in-vitro may give us clues as to their potential functions in vivo. For instance, the abilities of G&#981; to consume large amounts of neutrophil granules and debris, the presence of large vacuoles, and the expression LC3B - an autophagy protein which contributes to diminishing inflammation through regulatory interactions with innate immune signaling pathways,22 - all of which support scavenging abilities. As such, these findings also indicate that G&#981; might be functioning at inflammatory sites where the M&#981;/DC system is insufficient or overwhelmed, and thus contribute to the resolution of inflammation. This notion might be supported by the fact that in mixed monocyte/neutrophil cultures G&#981; development is hampered.14 Also, given that G&#981; express oxLDL scavenger receptors (CD36, CD68), internalize oxLDL, and produce ROS in response to it, may indicate that they are involved in atherosclerotic processes to resolve inflammation. Since G&#981; developed only from neutrophils which migrated towards IL-8, and neutrophils&#39; transmigration across endothelial monolayers towards IL-8 represents neutrophil recruitment to acute inflammatory sites, this finding also may support anti-inflammatory functions. Conversely, the performance of G&#981; in certain inflammatory conditions might enable them to discharge granule constituents and ROS, thus, contributing to persistent inflammation and tissue damage.20 However, overall, their autophagic abilities indicate that G&#981; are likely involved in diminishing the inflammatory response rather than perpetuating it.
Interestingly we have recently identified the presence of G&#981; in human atherosclerotic plaques. (in preparation). Yet, a great number of questions remain to be unraveled. For instance, are G&#981; pro- or anti-inflammatory? What are the factors which determine their formation and function in vitro or in vivo? Which specific neutrophil subpopulation is their precursor cell that facilitates their development into G&#981;? Are they associated with certain pathologies and which? Collectively, posing interesting questions as to their origin and potential functions.
However critical steps and pitfalls within the protocol should be kept in mind. A critical step in&#160;the development of G&#981; is culturing the pure neutrophils in medium devoid of cytokines, growth factors or antibiotics. Another critical step is to rule out that G&#981; develop from contaminating monocytes and to ascertain the neutrophilic origin of G&#981;. Thus, after blood separation by discontinuous gradient, the neutrophils were further subjected to an additional step of purification by flow cytometry using granulocyte gating and CD15+/CD14- markers. The developed G&#981; obtained from neutrophils that were further purified by flow cytometry separation did not differ from those that were not subjected to this step of purification. Therefore, most of the experiments were conducted without the flow cytometry step of purification due to additional cell loss. Of note, in some rare instances some eosinophils were noted in culture. Their size remained unchanged throughout the culture period. We should also note that although there are a number of methods for neutrophil separation from human blood, the method described here is the only method we employed and therefore we cannot compare G&#981; development by other available methods for neutrophil separation.
A major pitfall in investigating G&#981; results from the inability to obtain sufficient numbers of pure G&#981; population suitable for various biochemical assays. It is basically impossible in the conditions our experiments were conducted. First, the yield of G&#981; is low. From 1.0 x 106 PMN seeded about 100 - 200 G&#981; develop after seven days in culture, depending on the blood donor. Second, it is basically difficult at the moment to separate the developed G&#981; in culture from the remaining neutrophil debris in the dish. These limitations made it practically impossible to analyze the cells by biochemical or molecular biology methods. Therefore, this protocol is focused at describing G&#981; identification and function by using light and confocal microscopy. Their morphological transformation from neutrophils into G&#981; in culture was also followed by live cell imaging and time lapse microscopy.14 Apparently, much larger blood volumes may be needed in order to implement biochemical or molecular biology methods and overcome the low yield obtained and separating the viable G&#981; from neutrophils&#39; debris in the dish.
In summary, we have recently described for the first time the development of G&#981; in culture, a subpopulation of long-lived phagocytes of neutrophilic origin. Therefore, this is the only method currently available to obtain G&#981; in culture, although the two major limitations mentioned above should be overcome (the low yield of the G&#981; obtained in culture and the inability to separate the developed G&#981; from the neutrophil debris in the culture dish). Still, their preparation and identification, presented in this protocol, is essential for scientists interested in inflammatory responses and neutrophil biology and plasticity, in order to further investigate the potential significance and functions of G&#981;.

Polymorphonuclear neutrophils (PMN) constitute the largest population of leukocytes in the blood, serving as the first line of defense against invading pathogens by producing a wide range of cytotoxic molecules. The traditional view has long been that of blood circulating, short lived, professional phagocytes, which are the first to arrive to acute inflammatory sites to combat infections and aid in the clearance of pathogens and harmful particles.1 In their non-activated state, neutrophils are constitutively committed to apoptosis. When migrating from the blood to inflammatory sites, neutrophils undergo activation to resolve inflammation. They phagocytose and kill invading microorganisms, by producing an array of cytotoxic molecules as reactive oxygen species (ROS), lytic enzymes such as neutrophil elastase (NE) and cathepsins with potent microbicidal activity. In order to trap pathogens, neutrophils also release extracellular traps (NETs) which consist of nuclear chromatin threads containing antibacterial peptides and various lytic enzymes. However, uncontrolled release of these cytotoxic molecules from neutrophils may also perpetuate inflammatory responses and induce damage to surrounding tissues.2 Therefore, an effective clearance of apoptotic neutrophils by macrophages (M&#981;) and dendritic cells (DC) is crucial to resolve inflammation.3,4,5,6
In recent years however, it has become increasingly evident that neutrophils are highly versatile cells, whose functions go far beyond phagocytosis and pathogen killing.6,7 By undergoing priming or activation, neutrophil plasticity is gradually gaining attention. For instance, bacteria and mycobacteria challenged neutrophils were shown to secrete interleukin (IL)-10 and control the inflammatory response, suggesting the presence of immuno-regulatory responses.8 Post-mitotic neutrophils were shown to trans-differentiate into M&#981;-like cells, or DC-like cells by digesting and presenting antigen fragments when treated with cytokines and growth factors,9,10 thus, serving a critical role in integrating innate and adaptive responses.3,6 Activation by growth factors promoted engulfment of apoptotic neutrophils or cell debris, thereby, facilitating clearance of debris at inflammatory sites and the resolution of inflammation,3,9 particularly when the M&#981;/DC clearance system is insufficient or overwhelmed,11,12 suggesting potential &#39;self-regulation&#39; to help resolve the inflammatory response. This, since apoptosis is a form of regulated self-death which can inhibit the extracellular release of cytotoxic compounds and thus prevent injury to surrounding tissues.6
Prolonged survival is another feature of neutrophil activation and was demonstrated by treatment with various host derived factors such as granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), inflammatory cytokines such as interferon (IFN)-&#947;, tumor necrosis factor (TNF)-&#945; and/or pathogen derived products, thus, allowing neutrophils to modulate their survival response.6 In fact, neutrophil survival is a prerequisite for its plasticity and was associated with its ability to perform phagocytosis.6,13 Accordingly, it was also shown to associate with phenotypic and functional changes which depended on upregulated gene expression by inducing the synthesis of new proteins involved in neutrophil lifespan extension, and diminished apoptosis.10
Unlike neutrophils which are short lived and constitutively undergo apoptosis in culture, or the cytokines/growth factors-activated neutrophils, described above, which have extended life span, we have recently identified a new, small subpopulation of neutrophils that develops spontaneously in prolonged standard culture conditions from freshly isolated human blood neutrophils without externally adding cytokines or growth factors.14 These neutrophil-derived cells, which were not described before in the literature were termed giant phagocytes (G&#981;). The G&#981; have extended lifespan in culture, they are fully developed within 5-7 days, and are characterized by unique morphological features, phenotypic expression and functions. They are vastly enlarged due to autophagocytosis of dead neutrophil remnants, vacuolated, and contain phagolysosomes. The G&#981; express the specific neutrophil granules marker - cluster of differentiation (CD)66b, the azurophilic granules markers &#8211; CD63 and myeloperoxidase (MPO) and additional neutrophils markers such as CD11b, NE, CD15, the NADPH oxidase subunits gp91-phox and p22-phox, and the autophagy marker -LC3BII.14,15 Functionally, they actively take-up latex beads and zymosan particles, and generate ROS in response to zymosan and phorbol 12-myristate 13-acetate (PMA) stimulation. Interestingly, unlike fresh neutrophils, G&#981; also intensively express the scavenger receptors CD68 and CD36, take-up oxidized low density lipoprotein (oxLDL), and generate ROS in response to stimulation with oxLDL. Additionally, G&#981; are devoid of the monocytic lineage markers CD14, CD16 and CD163 or the dendritic markers CD1c and CD141. Moreover, phagocytosis and autophagy and likely functional NADPH oxidase are prerequisites for their development. This since, the phagocytosis-inhibitor cytochalsin B, the autophagy inhibitors 3-methyladenine (3-MA) and bafilomycin (BafA1) and the NADPH oxidase inhibitor - diphenylene iodonium (DPI) &#8211; prevented their development. Additionally, monocytes/neutrophils co-cultures as well as exposure to intermittent hypoxia hampered their development, whereas neutrophil adaptation to sustained hypoxia was evident.14,15 Their suggested development in culture is illustrated in Figure 1.The protocol in the present paper describes step by step the preparation of G&#981; from freshly isolated circulating human blood neutrophils, their development, identification and some basic characteristics. This protocol can be used to further investigate and reveal the broad spectrum and the roles of these newly described and intriguing neutrophil-derived G&#981; in order to characterize their significance and their potential functions.
<img alt="Figure 1" src="/files/ftp_upload/54826/54826fig1.jpg" /> 
Figure 1: Schematic Representation of Giant Cells Development in 7 Day Neutrophil Cultures. It is suggested that at inflammatory sites (1) neutrophils undergo apoptotic cell death, and (2) release membrane-encircled fragments containing nuclear debris, granules (green and red dots), and other subcellular constituents which trigger autophagy mechanisms. (3) Giant phagocytes (G&#981;) develop in long-term neutrophil cultures devoid of cytokines or growth factors by internalizing apoptotic bodies and neutrophil debris, while maintaining functional NADPH oxidase.They are characterized by various neutrophilic CD66b+/CD63+/MPO+/ CD15+/CD11b+/NE markers, large phagosomes enclosing granules and cell debris, and scavenger receptors CD36 and CD68. G&#981; are mostly mononucleated cells, capable of internalizing also various particles and oxidized LDL and generate ROS. The membranes of the vacuoles filling G&#981; contain LC3B (marked in dark blue), a marker of autophagosomal membrane, suggesting a strict association between autophagy and giant phagocyte formation. G&#981; do not develop in medium containing GM-CSF/IL-4. Also, inhibitors such as the NADPH oxidase inhibitor &#8211; diphenylene iodonium (DPI), the autophagy inhibitors 3-methyladenine (3-MA) and bafilomycin (BafA1) and the phagocytosis inhibitor cytochalasin B (Cyto. B) abolish their formation. (4) Potential G&#981; functions in vivo may include anti- or pro-inflammatory properties and participation in atherosclerotic processes (this figure is based on our findings14,15 and was modified from the accompanying Editorial by Berton20). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54826/54826fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>Sterile scalp vein set (21GX3/4)&amp;nbsp;</td><td>Bio Diagnostics Ltd.</td><td># 20080312</td><td>A strile needle for venipancture</td></tr><tr><td>VACUETTE HOLDEX Single-Use Holder PP</td><td>Greiner Bio-One</td><td># 450263</td><td>For securing during venipuncture&amp;nbsp;</td></tr><tr><td>VACUETTE Tube K3E K3EDTA (16 x 100/9 mL)</td><td>Greiner Bio-One</td><td># 455036</td><td>Sterile tube for blood collection</td></tr><tr><td>Nunclon MultiDish (24 well x 1 ml)</td><td>Thermo Scientific</td><td># 142475</td><td></td></tr><tr><td>Polypropylene conical centrifuge tube (50 ml)</td><td>Greiner Bio-One</td><td># E14103PJ</td><td></td></tr><tr><td>Transwell-24 well (transmigration assay)</td><td>Corning</td><td># CA-3415</td><td>Polycarbonate membrane, 6.5 mm diameter, 3 &amp;mu;m pores)&amp;nbsp;</td></tr><tr><td>RPMI-1640 medium</td><td>BioIndustries</td><td># 01-100-1A</td><td>Do not add antibiotics&amp;nbsp;&amp;nbsp;</td></tr><tr><td>EA.hy926 (ATCC CRL2922)&amp;nbsp;&amp;nbsp;</td><td>BioIndustries</td><td># CRL-2922</td><td></td></tr><tr><td>ATCC-formulated Dulbecco&amp;rsquo;s&amp;nbsp; Modified Eagle&amp;rsquo;s Medium</td><td>BioIndustries</td><td># 302002</td><td>complete growth medium</td></tr><tr><td>Polysucrose - Histopaque1119</td><td>Sigma-Aldrich</td><td># 1119-1</td><td>Tissue culture grade</td></tr><tr><td>Polysucrose - Histopaque1077</td><td>Sigma-Aldrich</td><td># 1077-1</td><td>Tissue culture grade&amp;nbsp;</td></tr><tr><td>Phosphate buffered saline (PBS) - ion free</td><td>BioIndustries</td><td># 02-023-1A</td><td>Cell biology and molecular biology grade</td></tr><tr><td>Heat inactivated Fetal calf serum (HI-FCS)</td><td>BioIndustries</td><td># 04-121-1B</td><td>Cell biology grade or tissue culture grade, low LPS</td></tr><tr><td>NaCl</td><td>Sigma</td><td># S3014</td><td>Molecular biology grade, suitable for cell culture</td></tr><tr><td>Paraformaldehyde, 16%</td><td>Electron Microscopy Sciences</td><td># 15710</td><td>Cell bology grade or tissue culture grade (only 16% PFA)</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td># 9002-93-1</td><td>Molecular biology grade&amp;nbsp;</td></tr><tr><td>Normal Goat Serum</td><td>BioIndustries</td><td># 04-009-1</td><td>Cell biology and molecular biology grade</td></tr><tr><td>Trypan blue</td><td>BioIndustries</td><td># 031021B</td><td>Tissue culture grade&amp;nbsp;</td></tr><tr><td>May-Gr&amp;uuml;nwald</td><td>Sigma-Aldrich</td><td># MG500</td><td>Cell biology grade-(procedure No GS-10)</td></tr><tr><td>Giemsa stain Kit</td><td>Sigma</td><td># 48900</td><td>Cell biololgy grade-(procedure No GS-10)</td></tr><tr><td>Fibronectin</td><td>BioIndustries</td><td># 03090105</td><td></td></tr><tr><td>Human Interleukin-8 (CXCL8)</td><td>PeproTech</td><td># 200-08-5</td><td></td></tr><tr><td>Anti-CD14 (clone 5A3B11B5)</td><td>Santa Cruz Biotechnologies</td><td># sc-58951</td><td>Mouse IgG2b; expressed by&amp;nbsp; monocytes</td></tr><tr><td>Anti-CD63 (clone MX-49.129.5)</td><td>Santa Cruz Biotechnologies</td><td># sc-5275</td><td>Mouse IgG1; expressed by neutrophils</td></tr><tr><td>Anti-CD66b (clone 80H3)</td><td>AbD Serotec</td><td># MCA216</td><td>Mouse IgG1; expressed by neutrophils</td></tr><tr><td>Anti-CD1c (BDCA-1) (clone AD5-8E7)</td><td>MACS Miltenyi Biotec</td><td># 130-090-695</td><td>Mouse IgG2a; expressed by&amp;nbsp; dendritic cells</td></tr><tr><td>Anti-CD15 (clone MY-1)</td><td>Abcam</td><td># ab754</td><td>Mouse IgM; expressed by neutrophils</td></tr><tr><td>Anti-Cytochrome b-245 Light Chain (p22-phox) (clone 44.1)</td><td>BioLegend</td><td># 650001</td><td>Mouse IgG2a; to recognize neutrophil NADPH oxidase complex</td></tr><tr><td>Anti-CD68</td><td>Protein Tech</td><td># 16192-1-AP</td><td>Rabbit IgG; to recognize oxLDL scavenger receptor&amp;nbsp;</td></tr><tr><td>Anti-LC3B</td><td>Sigma</td><td># L7543</td><td>Rabbit IgG&amp;nbsp;</td></tr><tr><td>Anti-Myeloperoxidase</td><td>Abcam</td><td># ab45977</td><td>Rabbit IgG</td></tr><tr><td>Anti-Neutrophil elastase</td><td>Calbiochem</td><td># 481001</td><td>Rabbit IgG&amp;nbsp;</td></tr><tr><td>Anti-NOX2 (gp91-phox)</td><td>Abcam</td><td># ab131083</td><td>Rabbit IgG</td></tr><tr><td>Anti-CD36 (SR-B3)</td><td>Novus Biologicals</td><td># NB400-144</td><td>Rabbit IgG</td></tr><tr><td>Purified Mouse IgG1, &amp;kappa; Isotype Control (clone MG1-45)</td><td>BioLegend</td><td># 401401</td><td>Antibody used as isotype control</td></tr><tr><td>Purified Mouse IgG2a, &amp;kappa; Isotype Control (clone MOPC-173)</td><td>BioLegend</td><td># 400263</td><td>Antibody used as isotype control</td></tr><tr><td>Normal rabbit IgG</td><td>Santa Cruz Biotechnologies</td><td># sc-2027</td><td>Antibody used as isotype control</td></tr><tr><td>CF488A Goat Anti-Rabbit IgG (H+L)</td><td>Biotium&amp;nbsp;</td><td># 20012</td><td>Anti-Rabbit IgG with the green fluorescent dye CF488A</td></tr><tr><td>CF647 Goat Anti-Rabbit IgG (H+L)</td><td>Biotium&amp;nbsp;</td><td># 20043</td><td>Anti-Rabbit IgG with the red fluorescent dye CF647</td></tr><tr><td>CF488A Goat Anti-Mouse IgG (H+L)</td><td>Biotium&amp;nbsp;</td><td># 20010</td><td>Anti-Mouse IgG with the green fluorescent dye CF488A</td></tr><tr><td>CF647 Goat Anti-Mouse IgG (H+L)</td><td>Biotium&amp;nbsp;</td><td># 20040</td><td>Anti-Mouse IgG with the red fluorescent dye CF647</td></tr><tr><td>Fluorescent Mounting Medium with DAPI</td><td>Vectashield H-1000; Vector Lab Inc.</td><td># E19-18</td><td>Nuclear staining</td></tr><tr><td>Confocal laser scanning microscope (LSM 700)&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Carl Zeiss</td><td>Ser.# 3523000380</td><td>Plan Apo 40X immersion oil objective&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Zeiss CLSM software (ZEN 2010)</td><td>Carl Zeiss MicroImaging GmbH</td><td>version 6.0</td><td>For colocalization analysis</td></tr><tr><td>ImageJ software</td><td>Wayne Rasband, NIH, USA</td><td>version 1.49k</td><td>For determination of cell&amp;nbsp; areas and fluorescence intensity</td></tr><tr><td>Light microscope (Axiovert 25)</td><td>Carl Zeiss</td><td>Ser.# 201060153</td><td>Examination of cells in culture</td></tr><tr><td>Centrifuge (Megafuge 1.0 R)</td><td>Heraeus Instruments</td><td># D-37520</td><td>Cells separation from blood; cytospins preparation</td></tr><tr><td>Inverted fluorescent microscope (Zeiss Axio Observer Z.1)</td><td>Carl Zeiss</td><td>Ser.# 3834001470</td><td>Demonstration of giant phagocytes development</td></tr><tr><td>Temperature-controlled incubation system (Cube&amp;amp;Box)</td><td>Life Imaging Services</td><td></td><td>Temperature control system for microscopes</td></tr><tr><td>High&amp;nbsp;resolution digital CCD camera (AxioCam HRm)</td><td>Carl Zeiss</td><td>Ser.# 117090279</td><td>For capturing high-contrast&amp;nbsp; image data from an examined cell objects</td></tr><tr><td>Hydrophobic barrier pen (Elite PAP Pen)</td><td>Diagnostic Biosystems</td><td># K039</td><td>For defining cell perimeter for immunoflourescence staining</td></tr></tbody></table>

development and identification of a novel subpopulation of human neutrophil-derived giant phagocytes in vitro

Neutrophils (PMN) are best known for their phagocytic functions against invading pathogens and microorganisms. They have the shortest half-life amongst leukocytes and in their non-activated state are constitutively committed to apoptosis. When recruited to inflammatory sites to resolve inflammation, they produce an array of cytotoxic molecules with potent antimicrobial killing. Yet, when these powerful cytotoxic molecules are released in an uncontrolled manner they can damage surrounding tissues. In recent years however, neutrophil versatility is increasingly evidenced, by demonstrating plasticity and immunoregulatory functions. We have recently identified a new neutrophil-derived subpopulation, which develops spontaneously in standard culture conditions without the addition of cytokines/growth factors such as granulocyte colony-stimulating factor (GM-CSF)/interleukin (IL)-4. Their phagocytic abilities of neutrophil remnants largely contribute to increase their size immensely; therefore they were termed giant phagocytes (G&#981;). Unlike neutrophils, G&#981; are long lived in culture. They express the cluster of differentiation (CD) neutrophil markers CD66b/CD63/CD15/CD11b/myeloperoxidase (MPO)/neutrophil elastase (NE), and are devoid of the monocytic lineage markers CD14/CD16/CD163 and the dendritic CD1c/CD141 markers. They also take-up latex and zymosan, and respond by oxidative burst to stimulation with opsonized-zymosan and PMA. G&#981; also express the scavenger receptors CD68/CD36, and unlike neutrophils, internalize oxidized-low density lipoprotein (oxLDL). Moreover, unlike fresh neutrophils, or cultured monocytes, they respond to oxLDL uptake by increased reactive oxygen species (ROS) production. Additionally, these phagocytes contain microtubule-associated protein-1 light chain 3B (LC3B) coated vacuoles, indicating the activation of autophagy. Using specific inhibitors it is evident that both phagocytosis and autophagy are prerequisites for their development and likely NADPH oxidase dependent ROS. We describe here a method for the preparation of this new subpopulation of long-lived, neutrophil-derived phagocytic cells in culture, their identification and their currently known characteristics. This protocol is essential for obtaining and characterizing G&#981; in order to further investigate their significance and functions.

Neutrophil Autophagocytosis and Development in Culture
Neutrophil autophagocytosis and their development into G&#981; within 7 days in culture is shown in Figures 3 and 4. By days 4 - 7, their size was vastly enlarged,15 and autophagosytosis was evident as early as 90 min after co-culturing the neutrophils with fluorescent membrane stains (PKH-26, red; PKH-67, green).14 As a control to this neutrophil subpopulation, some neutrophil cultures were also treated with GM-CSF/IL-4. The cytokine-treated cells increased in size within 7 - 14 days in culture as previously described.18,19 But, were smaller than G&#981; and had cytoplasmic projections resembling DC-like cells (Figure 5), as reported previously by Oehler et al.19 Also, the GM-CSF/IL-4 treated cells were negative or had a low CD66b expression,15 clearly demonstrating morphological and potentially functional differences as well.
<img alt="Figure 3" src="/files/ftp_upload/54826/54826fig3.jpg" /> 
Figure 3: Autophagocytosis in the Developing Giant Phagocytes (G&#981;) in Culture. Freshly isolated purified neutrophils were labeled with PKH-67 (green) or PKH-26 (red) membrane fluorescent dyes at zero time, and then co-cultured and followed up to seven days. Cells were spun onto glass slides, nuclei were stained with DAPI and samples were analyzed by confocal microscopy. Autophagocytosis is already noticeable after 90 min of co-culture. Merging of red and green into yellow and orange is clearly evident in the developing G&#981;. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54826/54826fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54826/54826fig4.jpg" /> 
Figure 4: Development of Giant Phagocytes (G&#981;) in Culture. Freshly isolated purified neutrophils were followed up to 7 days in culture. Cells were spun onto glass slides at the indicated time intervals, stained with May Grunewald-Giemsa, and analyzed with a bright field microscopy. An individual with few eosinophils is presented for comparison. Note that the size of eosinophils remains unchanged in culture. Magnification 100X oil. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54826/54826fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/54826/54826fig5.jpg" /> 
Figure 5: Comparison Between the Development of Giant Phagocytes (G&#981;) and GM-CSF/IL Treated Neutrophils in Culture. (A) May Grunwald-Giemsa stained neutrophils cultured without (G&#981;) and with GM-CSF/IL-4 for 7 days. Samples were analyzed with a bright-field microscopy. Magnification, X40. Cells developed in cultures with medium supplemented with GM-CSF/IL-4 show widespread cytoplasmic projections but are smaller than G&#981;. (B) Freshly isolated neutrophils were labeled with PKH-26 (red) dye and cultured in cytokine-free medium for 7 days or labeled with PKH-67 (green) dye and cultured in medium supplemented with GM-CSF/IL-4 for 7 days. Then, the developed cells were mixed in a 1:1 ratio and co-cultured for 2 hr. Cells were fixed and analyzed by confocal microscopy. This figure has been modified from reference.15 <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54826/54826fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
To further investigate the course of G&#981; development, their morphologic changes were also followed by time-lapse microscopy. Video-1 (day 3 to day 4) and video-2 (day 4 to day 5) demonstrate their development in purified neutrophil cultures. These G&#981; are non-adherent or lightly adherent with limited movement capacity and actively ingest surrounding neutrophil remnants and debris. In video-3, the movement of monocyte-derived M&#981; and G&#981; is compared in a mixed monocyte/neutrophil culture. The M&#981; actively crawls (left, unlabeled cell). The G&#981; (right), is bright PKH-26 labeled cell.
<img alt="Figure 1" src="/files/ftp_upload/54826/54826mov1.jpg" /> 
Video-1: Demonstrates the Development of Giant Phagocytes in Purified PMN Cultures on Days 3 - 4 by Time-lapse Microscopy. Neutrophils were followed-up in culture from day 3 to day 4 by time-lapse microscopy.The time-lapse microscopy system is composed of inverted motorized fluorescent microscope, and a high resolution B/W CCD camera, with an on stage incubator. Image capture acquisition of time-lapse was taken every 10 min. Originally published in reference14 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54826/Video-1.mpg" target="_blank">Please click here to view this video.</a>
<img alt="Figure 1" src="/files/ftp_upload/54826/54826mov2.jpg" /> 
Video-2: Demonstrates the Development of Giant Phagocytes in Purified PMN Culture on Days 4 - 5 by Time-lapse Microscopy. Neutrophils were followed-up in culture from day 4 to day 5 by time-lapse microscopy. The time-lapse microscopy system is composed of inverted motorized fluorescent microscope, and a high resolution B/W CCD camera, with an on stage incubator. Image capture acquisition of time-lapse was taken every 10 min. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54826/Video-2.mp4" target="_blank">Please click here to view this video.</a>
<img alt="Figure 1" src="/files/ftp_upload/54826/54826mov3.jpg" /> 
Video-3: A Giant Phagocyte and a Macrophage Developed in Co-culture. Monocytes/neutrophils co-culture was followed-up from day 4 to day 5 by time-lapse microscopy. Monocyte-derived macrophage (left); bright (PKH-26 stained cell) neutrophil-derived giant phagocyte (right). The time-lapse microscopy system for video is composed of inverted motorized fluorescent microscope, and a high resolution B/W CCD camera, with an on stage incubator. Image capture acquisition of time-lapse was taken every 10 min. Originally published in reference14 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54826/Video-3.mp4" target="_blank">Please click here to view this video.</a>
Expression of Markers in Giant Phagocytes
The neutrophilic origin of G&#981; was verified by positive expression of the following neutrophil markers CD66b/CD63/MPO/NE/CD15 (Figure 6). The G&#981; also expressed NADPH oxidase, the oxLDL scavenger receptors &#8211; CD68 and CD36, and contained LC3B-coated vacuoles and aggregates (identified by Western blotting as LC3BII15), demonstrating the presence of an autophagy marker. However they were negative for monocytic lineage (CD14, CD16 and CD163) and dendritic cells (CD1c and CD141) markers, suggesting that G&#981; did not arise from contaminating monocytes.
<img alt="Figure 6" src="/files/ftp_upload/54826/54826fig6.jpg" /> 
Figure 6: Expression of Various Markers for Neutrophils, Monocytes and Dendritic Cells in Giant Phagocytes (G&#981;) after 7 Days in Culture. Positive expression of the neutrophil specific granule marker CD66b, the azurophil granules markers CD63 and MPO, neutrophil elastase and CD15. Negative expression for the dendritic CD1c and CD141 markers and monocytic lineage markers CD14, CD16 and CD163. Additionally, G&#981; expressed the autophagy marker LC3B, the scavenger receptors CD68 and CD36 and the NADPH oxidase subunits gp91-phox/p22-phox. Nuclei were stained with DAPI, and samples were analyzed by confocal microscopy. This figure has been modified from references.14,15 <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54826/54826fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Functions of G&#981; - NADPH Oxidase Activation, ROS Production and Phagocytosis:
Phagocytosis of latex beads and opsonized zymosan was evident in G&#981;. G&#981; also generated basal ROS (Figure 7A), and responded to zymosan and PMA stimulation by oxidative burst (Figure 7B-D). However, unlike monocytes or neutrophils, G&#981; generated ROS also in response to oxLDL stimulation and were stained by Oil Red O (Figure 7B, F). Of note, treatment of fresh neutrophils with the NADPH oxidase inhibitor &#8211; DPI, not only inhibited ROS production, but also prevented G&#981; formation in culture, suggesting that ROS signaling is essential for G&#981; formation.14,15
<img alt="Figure 7" src="/files/ftp_upload/54826/54826fig7.jpg" /> 
Figure 7: Oxidative Burst, Phagocytosis, and oxLDL Uptake by Giant Phagocytes (G&#981;). (A) Basal ROS production is evident in lysosomes of G&#981;. (B) ROS production in response to oxidized LDL (oxLDL), PMA and zymosan (zymosan particles are clearly noted). (C) Nitroblue tetrazolium (NBT) test in G&#981; showing respiratory burst activity without and with PMA (slides are unstained, but the inserts are stained with May Grunwald-Giemsa). (D) NBT test and May Grunewald-Giemsa stained G&#981; with PMA and PMA/DPI which inhibited NADPH oxidase and ROS. (E) Phagocytosis of Latex and IgG-opsonized zymosan in PKH-26 (red) stained cells. (F) Oil Red O staining in untreated and oxLDL treated G&#981;. This figure has been modified from references.14,15 <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54826/54826fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Transmigration of PMN Across Endothelial Cells
In order to identify potential neutrophils sub-populations that might develop into G&#981;, the migration of neutrophils through endothelial cell monolayers was determined (Figure 8A). After 90 min, 62.3 &#177; 12.2% of the neutrophils transmigrated through endothelial cells towards IL-8 in the lower compartment. Of note, G&#981; positive for CD66b/CD15/LC3B developed only from the transmigrated population of neutrophils whereas the cells which developed from the non-migrating neutrophils fraction were smaller in size and negative for the neutrophilic markers CD66b/CD15 (Figure 8B, 8C).
<img alt="Figure 8" src="/files/ftp_upload/54826/54826fig8.jpg" /> 
Figure 8: Effects of IL-8-dependent PMN Transmigration Through Endothelial Cells on Giant Phagocyte (G&#981;) Formation. (A) A scheme illustrating neutrophil transmigration assay across endothelial cell monolayers (ECs) towards IL-8. This assay can be considered as a model for neutrophils recruitment to acute inflammatory sites. (B-C) In the cell migration assay (specified in protocol 3), transmigrating (B) and non-migrating (C) neutrophils fractions were cultured for seven days without growth factors (as in protocol 1). Then, cells were spun onto glass slides and analyzed by confocal microscopy. Fixed cells were stained for CD66b (red), LC3B (green) and CD15 (red). Nuclei were stained with DAPI (blue). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54826/54826fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro at JoVE.com

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

We describe here a method for obtaining and identifying a newly characterized subpopulation of neutrophil-derived giant phagocytes. These cells develop in culture from fresh human blood neutrophils, and are characterized by phagocytosis, autophagy, immensely large size, and extended lifespan. This method is essential to further investigate this unique neutrophil-derived subpopulation.

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

Neutrophils (PMN) are best known for their phagocytic functions against invading pathogens and microorganisms. They have the shortest half-life amongst ...

development-identification-novel-subpopulation-human-neutrophil

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

15.2K Views.  Technion-Israel Insitute of Technology.  We beschrijven hier een werkwijze voor het verkrijgen en identificeren van een nieuw kenmerk subpopulatie van neutrofielen afgeleide reus fagocyten. Deze cellen ontwikkelen in de cultuur van verse menselijk bloed neutrofielen, en worden gekenmerkt door fagocytose, autofagie, immens groot formaat, en verlengde levensduur. Deze methode is van essentieel belang om verder te onderzoeken deze unieke neutrofielen afgeleid subpopulatie. 

Video: Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

Direct Observation of Phagocytosis and NET-formation by Neutrophils in Infected Lungs using 2-photon Microscopy

After the gastrointestinal tract, the lung is the second largest surface for interaction between the vertebrate body and the 
environment. Here, an effective gas exchange must be maintained, while at the same time avoiding infection by the multiple pathogens that are inhaled 
during normal breathing. To achieve this, a superb set of defense strategies combining humoral and cellular immune mechanisms exists. One of the most 
effective measures for acute defense of the lung is the recruitment of neutrophils, which either phagocytose the inhaled pathogens or kill them by 
releasing cytotoxic chemicals. A recent addition to the arsenal of neutrophils is their explosive release of extracellular DNA-NETs by which bacteria 
or fungi can be caught or inactivated even after the NET releasing cells have died. We present here a method that allows one to directly observe neutrophils, 
migrating within a recently infected lung, phagocytosing fungal pathogens as well as visualize the extensive NETs that they have produced throughout the 
infected tissue. The method describes the preparation of thick viable lung slices 7 hours after intratracheal infection of mice with conidia of the mold 
Aspergillus fumigatus and their examination by multicolor time-lapse 2-photon microscopy. This approach allows one to directly investigate antifungal 
defense in native lung tissue and thus opens a new avenue for the detailed investigation of pulmonary immunity.

We show, how to use 2-photon microscopy for the observation of the dynamics of neutrophil granulocytes in infected lungs while they phagocytose pathogens or produce neutrophil extracellular traps (NETs).

After the gastrointestinal tract, the lung is the second largest surface for interaction between the vertebrate body and the 
environment. Here, an ...

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.

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 ...

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 ...

Imaging the Neutrophil Phagosome and Cytoplasm Using a Ratiometric pH Indicator

Neutrophils are crucial to host innate defense and, consequently, constitute an important area of medical research. The phagosome, the intracellular compartment where the killing and digestion of engulfed particles take place, is the main arena for neutrophil pathogen killing that requires tight regulation. Phagosomal pH is one aspect that is carefully controlled, in turn regulating antimicrobial protease activity. Many fluorescent pH-sensitive dyes have been used to visualize the phagosomal environment. S-1 has several advantages over other pH-sensitive dyes, including its dual emission spectra, its resistance to photo-bleaching, and its high pKa. Using this method, we have demonstrated that the neutrophil phagosome is unusually alkaline in comparison to other phagocytes. By using different biochemical conjugations of the dye, the phagosome can be delineated from the cytoplasm so that changes in the size and shape of the phagosome can be assessed. This allows for further monitoring of ionic movement.

This manuscript describes a simple method to measure the phagosomal pH and area as well as the cytoplasmic pH of human and mouse neutrophils using the ratiometric indicator seminaphthorhodafluor (SNARF)-1, or S-1. This is achieved using live-cell confocal fluorescence microscopy and image analysis.

Neutrophils are crucial to host innate defense and, consequently, constitute an important area of medical research. The phagosome, the intracellular ...

Bioparticle Microarrays for Chemotactic and Molecular Analysis of Human Neutrophil Swarming in vitro

Neutrophil swarming is a cooperative process by which neutrophils seal off a site of infection and promote tissue reorganization. Swarming has classically been studied in vivo in animal models showing characteristic patterns of cell migration. However, in vivo models have several limitations, including intercellular mediators that are difficult to access and analyze, as well as the inability to directly analyze human neutrophils. Because of these limitations, there is a need for an in vitro platform that studies swarming with human neutrophils and provides easy access to the molecular signals generated during swarming. Here, a multistep microstamping process is used to generate a bioparticle microarray that stimulates swarming by mimicking an in vivo infection. The bioparticle microarray induces neutrophils to swarm in a controlled and stable manner. On the microarray, neutrophils increase in speed and form stable swarms around bioparticle clusters. Additionally, supernatant generated by the neutrophils was analyzed and 16 proteins were discovered to have been differentially expressed over the course of swarming. This in vitro swarming platform facilitates direct analysis of neutrophil migration and protein release in a reproducible, spatially controlled manner.

This protocol generates bioparticle microarrays that provide spatially controlled neutrophil swarming. It provides easy access to the mediators that neutrophils release during migration and allows for quantitative imaging analysis.

Neutrophil swarming is a cooperative process by which neutrophils seal off a site of infection and promote tissue reorganization. Swarming has classically ...

Bioengineering

In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy

In response to invading pathogens, neutrophils release neutrophil extracellular traps (NETs), which are extracellular networks of DNA decorated with histones and antimicrobial proteins. Excessive NET formation (NETosis) and citH3 release during sepsis is associated with multiple organ dysfunction and mortality in mice and humans but its implications in dogs are unknown. Herein, we describe a technique to isolate canine neutrophils from whole blood for observation and quantification of NETosis. Leukocyte-rich plasma, generated by dextran sedimentation, is separated by commercially available density gradient separation media and granulocytes collected for cell count and viability testing. To observe real-time NETosis in live neutrophils, cell permeant and cell impermeant fluorescent nucleic acid stains are added to neutrophils activated either by lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate (PMA). Changes in nuclear morphology and NET formation are observed over time by fluorescence microscopy. In vitro NETosis is further characterized by co-colocalization of cell-free DNA (cfDNA), myeloperoxidase (MPO) and citrullinated histone H3 (citH3) using a modified double-immunolabelling protocol. To objectively quantify NET formation and citH3 expression using fluorescence microscopy, NETs and citH3-positive cells are quantified in a blinded manner using available software. This technique is a specific assay to evaluate the in vitro capacity of canine neutrophils to undergo NETosis.

In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy

We describe methods to isolate canine neutrophils from whole blood and visualize NET formation in live neutrophils using fluorescence microscopy. Also described are protocols to quantify NET formation and citrullinated histone H3 (citH3) expression using immunofluorescence microscopy.

In response to invading pathogens, neutrophils release neutrophil extracellular traps (NETs), which are extracellular networks of DNA decorated with ...

Quantifying NET Formation Using Dual-Dye Live Cell Analysis

Real-Time High Throughput Technique to Quantify Neutrophil Extracellular Traps Formation in Human Neutrophils

Neutrophils are myeloid-lineage cells that form a crucial part of the innate immune system. The past decade has revealed additional key roles that neutrophils play in the pathogenesis of cancer, autoimmune diseases, and various acute and chronic inflammatory conditions by contributing to the initiation and perpetuation of immune dysregulation through multiple mechanisms, including the formation of neutrophil extracellular traps (NETs), which are structures crucial in antimicrobial defense. Limitations in techniques to quantify NET formation in an unbiased, reproducible, and efficient way have restricted our ability to further understand the role of neutrophils in health and diseases. We describe an automated, real-time, high-throughput method to quantify neutrophils undergoing NET formation using a live cell imaging platform coupled with a membrane permeability-dependent dual-dye approach using two different DNA dyes to image intracellular and extracellular DNA. This methodology is able to help assess neutrophil physiology and test molecules that can target NET formation.

Author Spotlight: Quantifying Neutrophil Extracellular Traps in Disease and Drug Screening Using Dual-Color Live-Cell Imaging

We present an automated high-throughput method to quantify neutrophil extracellular traps (NETs) utilizing the live cell analysis system, coupled with a membrane permeability-dependent dual-dye approach.

Neutrophils are myeloid-lineage cells that form a crucial part of the innate immune system. The past decade has revealed additional key roles that ...

Neutrophil Functional Assays for Screening Different Signaling Pathways

A Set of Screening Techniques for a Quick Overview of the Neutrophil Function

Neutrophils are known as one of the first lines of defense in the innate immune response and can perform many particular cellular functions, such as chemotaxis, reverse migration, phagocytosis, degranulation of cytotoxic enzymes and metabolites, and release of DNA as neutrophil extracellular traps (NETs). Neutrophils not only have tightly regulated signaling themselves, but also participate in the regulation of other components of the immune system. As fresh neutrophils are terminally differentiated, short-lived, and highly variable among individuals, it is important to make the most of the collected samples. Researchers often need to perform screening assays to assess an overview of the many neutrophil functions that may be affected by specific conditions under evaluation. A set of tests following a single isolation process of normal density neutrophils was developed to address this need, seeking a balance between speed, comprehensiveness, cost, and accuracy. The results can be used to reason and guide in-depth follow-up studies. This procedure can be carried out in an average time of 4 h and includes the evaluation of cell viability, reactive oxygen species (ROS) production, real-time migration, and phagocytosis of yeast on glass slides, leaving enough cells for more detailed approaches like omics studies. Moreover, the procedure includes a way to easily observe a preliminary suggestion of NETs after fast panoptic staining observed by light microscopy, with a lack of specific markers, albeit enough to indicate if further efforts in that way would be worthwhile. The diversity of functions tested combines common points among tests, reducing the analysis time and expenses. The procedure was named NeutroFun Screen, and although having&#160;limitations, it balances the aforementioned factors. Furthermore, the aim of this work is not a definite test set, but rather a guideline that can easily be adjusted to each lab&#39;s resources and demands.

Author Spotlight: Balancing Speed, Cost, and Accuracy in Neutrophil Function Assessment with NeutroFun Screen Protocol 

This protocol features a set of neutrophil functional assays to be used as a screening method to cover functions from different signaling pathways. The protocol includes an initial and simple evaluation of cell viability, purity, reactive oxygen species production, real-time migration, phagocytosis, and a preliminary suggestion of neutrophil extracellular traps.

Neutrophils are known as one of the first lines of defense in the innate immune response and can perform many particular cellular functions, such as ...

The MUB40 Peptide for Use in Detecting Neutrophil-Mediated Inflammation Events

Here, we provide a protocol involving the use of MUB40, a synthesized peptide with the ability to bind glycosylated lactoferrin stored at high concentrations in specific and tertiary granules of neutrophils. This protocol details how MUB40&#160;conjugated directly to a fluorophore can be used to stain neutrophils in fixed/permeabilized tissues as well as how this can be used in live-cell imaging to assay for neutrophil activation and de-granulation. Neutrophil detection methods are limited to species-specific monoclonal antibodies, which are not always suitable for certain applications. MUB40 does not penetrate the cell membrane and is thus excluded from lactoferrin stored in non-activated/non-permeabilized neutrophils. MUB40 has the added benefit of recognizing lactoferrin from a broad host range, making it especially useful for comparing results in studies involving multiple research models, reducing the number of duplicate reagents, and simplifying protocols through single-step staining.

The MUB40 Peptide for Use in Detecting Neutrophil-Mediated Inflammation Events

Here, we present a protocol to detect the presence of neutrophils in fixed/permeabilized histology sections and assess the activation state of live purified neutrophils. In particular, the MUB40&#160;peptide binds lactoferrin present in neutrophil-specific and tertiary granules. Exposure of the granule contents through either permeabilization or neutrophil activation allows for the marking of neutrophils.

Here, we provide a protocol involving the use of MUB40, a synthesized peptide with the ability to bind glycosylated lactoferrin stored at high ...

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 ...

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

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