We are grateful to Rafael Prados-Rosales for sharing the anti-MEV antisera and Navneet Dogra for performing nanoparticle tracking analysis.

1. Preparation of Iron-depleted Defined Medium
<ol>
	<li>Prepare 1 L of minimal medium (MM) by dissolving 5 g of KH2PO4, 5 g of L-asparagine, 20 mL of glycerol, and 2 g of dextrose in 900 mL of deionized water in a plastic container. Avoid glass to prevent iron contamination. Adjust the pH to 6.8 with 5 N NaOH and the volume to 1 L with water.</li>
	<li>Add 50 g of metal chelating resin (MCR) and gently agitate using a magnetic stir bar for 24 h at 4 &#176;C. Sterilize and remove the MCR by filtration through a 0.22 &#181;m filter unit with a plastic receiver. To accelerate filtration and prevent filter clogging, let the resin sediment for about 30 min before filtration. 
	NOTE: This medium contains less than 2 &#181;M residual iron, as determined by atomic absorption spectroscopy.</li>
	<li>Supplement MM with 0.5 mg/L of ZnCl2, 40 mg/L of MgSO4, and 0.1 mg/L of MnSO4. Separately, prepare concentrated stocks (1,000x) of each of the metal supplements in deionized water and sterilize by filtration before MM supplementation. Iron-depleted, metal supplemented MM will be referred here as low iron MM (LIMM).</li>
	<li>From a 50 mM stock of FeCl3 dissolved in 10 mM HCl, add 1 mL to 1 L of LIMM (50 &#181;M final concentration) to prepare high iron MM (HIMM).</li>
</ol>
2. Growing Mycobacteria in Iron-limited Conditions
<ol>
	<li>Thaw out 50 &#181;L of a frozen 15% glycerol stock of Mtb and streak an agar plate supplemented with 10% ADN enrichment (5 g/L albumin, 2 g/L dextrose, and 0.85 g/L sodium chloride, 0.2% glycerol and 0.05% Tween-80)10. Incubate the plate at 37 &#176;C until colonies are visible.</li>
	<li>Inoculate a single colony of Mtb10 in 2 mL of mycobacterial broth medium (Table of Materials) supplemented with ADN enrichment. Incubate with agitation at 37 &#176;C.</li>
	<li>Let the Mtb culture grow to the late logarithmic phase (OD540&#160;is ~0.8). To check this, measure the OD540 using a spectrophotometer.</li>
	<li>Spread 200 &#181;L of the late logarithmic culture onto the mycobacterial agar plates (Table of Materials) supplemented with 0.2% glycerol, 0.05% Tween-80, and ADN. Inoculate at least 5 plates. Incubate the plates at 37 &#176;C until bacterial growth is visible as a confluent layer. This takes ~1 week for Mtb.</li>
	<li>Wet a sterile cotton swab in LIMM. Use this swab to collect bacteria from the agar plates and inoculate 100 mL of LIMM to prepare a concentrated bacterial suspension with an OD540 of ~1.0.</li>
	<li>Dilute this suspension 10 times to 1 L with LIMM and divide it into two, 2 L sterile plastic bottles, each one containing 500 mL of culture.</li>
	<li>Take out 2 mL of culture and transfer it to a 5 mL culture tube. Add 10 &#181;L of 10% vol/vol tyloxapol.</li>
	<li>Incubate the cultures at 37 &#176;C standing for 14 days.</li>
</ol>
3. Collection of MEVs
<ol>
	<li>Measure the OD540 of the 2 mL culture at the time of collection. Make 1:10 serial dilutions of the culture and plate 100 &#181;L of each dilution on agar plates with ADN and 0.05% Tween-80.</li>
	<li>Transfer the culture to five 225 mL conical centrifuge tubes and centrifuge at 2,850 x g for 7 min at 20 &#176;C.</li>
	<li>Collect the culture supernatant with a 50 mL pipette and filter sterilize it through a 0.22 &#181;m filter unit.</li>
</ol>
4. Isolation of MEVs
<ol>
	<li>Transfer the culture filtrate into a stirred cell ultrafiltration system placed at 4 &#176;C and filter the concentrate at &#60;50 psi through a 100 kDa cutoff membrane to ~50 mL.</li>
	<li>Centrifuge the concentrated culture filtrate at 15,000 x g for 15 min at 4 &#176;C and collect the supernatant.</li>
	<li>Centrifuge the culture filtrate in polycarbonate ultracentrifugation tubes at 100,000 x g for 2 h at 4 &#176;C.</li>
	<li>Resuspend the membranous pellets in a total of 1 mL of sterile phosphate buffered saline (PBS) by gentle pipetting.</li>
	<li>Mix 0.5 mL of the pellet suspension obtained in step 4.4 with 1.5 mL of 60% iodixanol solution, yielding a final iodixanol concentration of 45% wt/vol. Dispense this mix at the bottom of a 13 mm x 51 mm polypropylene thin-walled ultracentrifuge tube.</li>
	<li>Overlay the MEV-iodixanol 45% suspension with 1 mL of 40%, 35%, 30%, 25%, and 20% (vol/vol in PBS) iodixanol solutions and 1 mL of PBS at the top.</li>
	<li>Centrifuge at 100,000 x g for 18 h at 4 &#176;C.</li>
	<li>Collect the 1 mL density gradient fractions starting from the top using a 1 mL Hamilton syringe.</li>
	<li>Dilute each collected fraction to 20 mL with PBS and centrifuge at 100,000 x g for 2 h at 4 &#176;C.</li>
	<li>Remove the supernatant and suspend the pellet in 0.5 mL of PBS. Store this pellet at 4 &#176;C.</li>
</ol>
5. Quantification of MEVs
<ol>
	<li>Measure the protein concentration in each fraction by a Bradford assay (Table of Materials), following the manufacturer&#39;s guidelines.</li>
	<li>Perform membrane lipid analysis.
	<ol>
		<li>Incubate 10 &#181;L of each gradient fraction with the fluorescent membrane probe 1-(4-Trimethylammoniumphenyl)-6-Phenyl-1,3,5-Hexatriene p-Toluenesulfonate (TMA-DPH) at a final concentration of 1 &#181;g/mL in a final 50 &#181;L volume of PBS in 96 well black plates.</li>
		<li>Incubate the plates at 33 &#176;C for 20 min.</li>
		<li>Measure the fluorescence at 360 nm excitation and 430 nm emission.</li>
	</ol>
	</li>
</ol>
6. Qualitative analysis of MEVs
<ol>
	<li>Perform protein electrophoresis.
	<ol>
		<li>Mix approximately 1 &#181;g of MEV samples in 16 &#181;L with 4 &#181;L of 5x sample loading buffer (10% w/v SDS, 10 mM dithiothreitol, 20% v/v glycerol 0.2 M Tris-HCl, pH 6.8 0.05% w/v bromophenol blue) and heat the samples in sample buffer at 85 &#176;C for 5 min.</li>
		<li>Load in a 10% Tris/Glycine SDS-polyacrylamide gel11 and run at 10 V/cm in running buffer (25 mM Tris base, 190 mM glycine, 0.1% SDS) until the blue dye front reaches the bottom of the gel.</li>
		<li>Stain the gel with an ultrasensitive protein staining solution (Table of Materials).</li>
	</ol>
	</li>
	<li>Perform the dot blot.
	<ol>
		<li>Load 2 &#181;L of a MEVs suspension with a concentration of approximately 0.5 &#181;g/&#181;L, and twofold serial dilutions on a nitrocellulose membrane and process for a dot blot according to the manufacturer&#39;s instructions.</li>
		<li>Use an antiserum raised in mice against a preparation of MEVs at a dilution of 1:5,000 as the primary antibody and a goat anti-mouse coupled to horseradish peroxidase (HRP) at a 1:10,000 dilution as the secondary antibody. Detect antigen-antibody complexes with an appropriate HRP substrate Blotting Detection Reagent and an imaging system.</li>
	</ol>
	</li>
	<li>Perform negative staining and electron microscopy.
	<ol>
		<li>Fix 250 &#181;L of MEVs with 2% glutaraldehyde in 0.1 M cacodylate at room temperature for 2 h and incubate overnight in 4% formaldehyde, 1% glutaraldehyde, and 0.1% PBS.</li>
		<li>Stain the fixed samples with 2% osmium tetroxide for 90 min.</li>
		<li>Serially dehydrate the sample in ethanol and embed in Spurr&#39;s epoxy resin.</li>
		<li>Observe the MEVs under a transmission electron microscope.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Prados-Rosales, R., 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=Mycobacteria+release+active+membrane+vesicles+that+modulate+immune+responses+in+a+TLR2-dependent+manner+in+mice.">Mycobacteria release active membrane vesicles that modulate immune responses in a TLR2-dependent manner in mice.</a> Journal of Clinical Investigation. 121, 1471-1483 (2011).</li><li>Gupta, S., Rodriguez, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mycobacterial+extracellular+vesicles+and+host+pathogen+interactions.">Mycobacterial extracellular vesicles and host pathogen interactions.</a> Pathogens and Disease. 76 (4), (2018).</li><li>Athman, J. 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=Bacterial+Membrane+Vesicles+Mediate+the+Release+of+Mycobacterium+tuberculosis+Lipoglycans+and+Lipoproteins+from+Infected+Macrophages.">Bacterial Membrane Vesicles Mediate the Release of Mycobacterium tuberculosis Lipoglycans and Lipoproteins from Infected Macrophages.</a> Journal of Immunology. 195, 1044-1053 (2015).</li><li>Athman, J. 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=Mycobacterium+tuberculosis+Membrane+Vesicles+Inhibit+T+Cell+Activation.">Mycobacterium tuberculosis Membrane Vesicles Inhibit T Cell Activation.</a> Journal of Immunology. 198, 2028-2037 (2017).</li><li>Rath, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+regulation+of+vesiculogenesis+and+immunomodulation+in+Mycobacterium+tuberculosis.">Genetic regulation of vesiculogenesis and immunomodulation in Mycobacterium tuberculosis.</a> Proceedings of the National Academy of Science U.S.A. 110, E4790-E4797 (2013).</li><li>White, D. W., Elliott, S. R., Odean, E., Bemis, L. T., Tischler, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mycobacterium+tuberculosis+Pst/SenX3-RegX3+Regulates+Membrane+Vesicle+Production+Independently+of+ESX-5+Activity.">Mycobacterium tuberculosis Pst/SenX3-RegX3 Regulates Membrane Vesicle Production Independently of ESX-5 Activity.</a> mBio. 9, pii 00778 (2018).</li><li>Dauros Singorenko, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+membrane+vesicles+from+prokaryotes:+a+technical+and+biological+comparison+reveals+heterogeneity.">Isolation of membrane vesicles from prokaryotes: a technical and biological comparison reveals heterogeneity.</a> Journal of Extracellular Vesicles. 6, 1324731 (2017).</li><li>Prados-Rosales, R., Brown, L., Casadevall, A., Montalvo-Quiros, S., Luque-Garcia, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+identification+of+membrane+vesicle-associated+proteins+in+Gram-positive+bacteria+and+mycobacteria.">Isolation and identification of membrane vesicle-associated proteins in Gram-positive bacteria and mycobacteria.</a> MethodsX. 1, 124-129 (2014).</li><li>Prados-Rosales, R., 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=Role+for+Mycobacterium+tuberculosis+membrane+vesicles+in+iron+acquistion.">Role for Mycobacterium tuberculosis membrane vesicles in iron acquistion.</a> Journal of Bacteriology. 196, 1250-1256 (2014).</li><li>Sanders, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aseptic+Laboratory+Techniques:+Plating+Methods.">Aseptic Laboratory Techniques: Plating Methods.</a> Journal of Visualized Experiments. 63, e3064 (2012).</li><li>Harlow, E., Lane, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Antibodies. A laboratory manual. , (1988).</li><li>Lotvall, 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=Minimal+experimental+requirements+for+definition+of+extracellular+vesicles+and+their+functions:+a+position+statement+from+the+International+Society+for+Extracellular+Vesicles.">Minimal experimental requirements for definition of extracellular vesicles and their functions: a position statement from the International Society for Extracellular Vesicles.</a> Journal of Extracellular Vesicles. 3, 26913 (2014).</li></ol>

The authors have no conflicts of interest.

Multiple methods to purify eukaryotic cell-derived exosomes have been developed12. In contrast, there is limited information on effective methods to purify bacteria-derived EVs7. Efficient isolation of Mtb-derived EVs needs to consider the intrinsic difficulties in growing this pathogenic mycobacterium. Mtb has a long division time (~24 h) and should be handled in biosafety level three (BSL-3) conditions. Therefore, it is important to optimize the efficiency of MEV isolatio...

Mycobacterial extracellular vesicles (MEVs) are membrane-bound nanoparticles, 60&#8722;300 nm in size, naturally released by fast- and slow-growing mycobacteria1. MEVs released by pathogenic mycobacteria constitute a mechanism to interact with the host via immunologically active proteins, lipids, and glycolipids secreted in a concentrated and protected manner2,3,4. To characterize MEVs and understand their biogenesis and functions, strict and efficient methods of vesicle purification and validation are crucial. Thus far, MEVs have been isolated from the culture filtrates of mycobacteria grown in an iron-rich medium1,5,6,7,8.
However, previous work demonstrated that iron limitation greatly stimulates vesicle release in Mtb,possibly to capture iron via mycobactin, a siderophore secreted in MEVs9. Although procedures for MEVs isolation from Mtb cultured in high iron medium have been described, an efficient methodology to obtain MEVs from low iron cultures has not been reported. Therefore, the goal of this method is to isolate, purify, and quantify MEVs obtained from low iron cultures so that they can be used for biochemical and functional assays and for the analysis of genetic determinants of vesicle production in mycobacteria.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Amicon stirred cell Model 108</td>
			<td>EMD Milipore</td>
			<td>UFSC40001</td>
			<td>Cell Ultrafiltration system</td>
		</tr>
		<tr>
			<td>BD Polypropilene 225 ml conical tubes</td>
			<td>Fisher</td>
			<td>05-538-61</td>
			<td>Conical centrifuge tubes</td>
		</tr>
		<tr>
			<td>Biomax 100-kDa cut-off ultrafiltration membrane</td>
			<td>EMD Milipore</td>
			<td>PBHK07610</td>
			<td>Ultrafiltration membrane</td>
		</tr>
		<tr>
			<td>Chelex-100 resin</td>
			<td>Bio-Rad</td>
			<td>142-2842</td>
			<td>Metal chelating resin</td>
		</tr>
		<tr>
			<td>Middlebrook 7H10 Agar</td>
			<td>BD Difco</td>
			<td>262710</td>
			<td>Mycobacterial Agar plates</td>
		</tr>
		<tr>
			<td>Middlebrook 7H9 Broth</td>
			<td>BD Difco</td>
			<td>271310</td>
			<td>Mycobacterial broth medium</td>
		</tr>
		<tr>
			<td>Nitro cellulose blotting membrane</td>
			<td>GE Healthcare</td>
			<td>10600001</td>
			<td>Blotting Membrane</td>
		</tr>
		<tr>
			<td>Optiprep</td>
			<td>Sigma</td>
			<td>D1556</td>
			<td>Iodixanol</td>
		</tr>
		<tr>
			<td>Polycarbonate ultra centrifugation tubes 25 x 89 mm</td>
			<td>Beckman Coulter</td>
			<td>355618</td>
			<td>Polycarbonate ultra centrifugation tubes 25 x 89 mm</td>
		</tr>
		<tr>
			<td>Polypropylene thin walled centrifuge tube 13x15 mm</td>
			<td>Beckman Coulter</td>
			<td>344059</td>
			<td>Polypropylene thin walled centrifuge tube 13x15 mm</td>
		</tr>
		<tr>
			<td>Protein Assay dye</td>
			<td>BioRad</td>
			<td>5000006</td>
			<td>Bradford Protein Staining</td>
		</tr>
		<tr>
			<td>SYPRO Ruby</td>
			<td>Molecular Probes</td>
			<td>S12000</td>
			<td>Ultrasensitive protein stain</td>
		</tr>
		<tr>
			<td>TMA-DPH</td>
			<td>Molecular Probes</td>
			<td>T204</td>
			<td>1-(4-Trimethylammoniumphenyl)-6-Phenyl-1,3,5-Hexatriene p-Toluenesulfonate</td>
		</tr>
		<tr>
			<td>Vacuum filtration flasks</td>
			<td>CellPro</td>
			<td>V50022</td>
			<td>Filter Unit</td>
		</tr>
	</tbody>
</table>

isolation and characterization of extracellular vesicles produced by iron-limited mycobacteria

Mycobacteria, including Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis, naturally release extracellular vesicles (EVs) containing immunologically active molecules. Knowledge regarding the molecular mechanisms of vesicle biogenesis, the content of the vesicles, and their functions at the pathogen-host interface is very limited. Addressing these questions requires rigorous procedures for isolation, purification, and validation of EVs. Previously, vesicle production was found to be enhanced when M. tuberculosis was exposed to iron restriction, a condition encountered by Mtb in the host environment. Presented here is a complete and detailed protocol to isolate and purify EVs from iron-deficient mycobacteria. Quantitative and qualitative methods are applied to validate purified EVs.

MEVs were purified by differential sedimentation in a density gradient (Figure 1, Figure 2). Under the conditions described, MEVs separated mostly in gradient fraction 3 (F3), which corresponds to 25% iodixanol. This conclusion is based on the detection of protein, membrane lipid, microscopic visualization of intact MEVs, nanoparticle size distribution, and positive reactivity with an antivesicle antiserum (F...

Watch this Scientific Journal Video about Isolation and Characterization of Extracellular Vesicles Produced by Iron-limited Mycobacteria at JoVE.com

Isolation and Characterization of Extracellular Vesicles Produced by Iron-limited Mycobacteria

Mycobacterium tuberculosis shows increased production and release of extracellular vesicles in response to low iron conditions. This work details a protocol for generating low iron conditions and methods for the purification and characterization of mycobacterial extracellular vesicles released in response to iron deficiency.

Mycobacteria, including Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis, naturally release extracellular vesicles (EVs) ...

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Immunology and Infection

7.2K Views.  Rutgers, The State University of New Jersey. Mycobacterium tuberculosis shows increased production and release of extracellular vesicles in response to low iron conditions. This work details a protocol for generating low iron conditions and methods for the purification and characterization of mycobacterial extracellular vesicles released in response to iron deficiency.

Video: Isolation and Characterization of Extracellular Vesicles Produced by Iron-limited Mycobacteria

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is initiated by interaction of the pathogen with intestinal epithelial cells. This interaction leads to disruption of intercellular structures such as tight junctions (TJ). TJ ensure sealing of the epithelial layer to separate host tissue from gut lumen. Recent studies provide evidence that disruption of TJ by the parasitic protein EhCPADH112 is a prerequisite for E. histolytica invasion that is accompanied by epithelial barrier dysfunction. Thus, the analysis of molecular mechanisms involved in TJ disassembly during E. histolytica invasion is of paramount importance to improve our understanding of amoebiasis pathogenesis. This article presents an easy model that allows the assessment of initial host-pathogen interactions and the parasite invasion potential. Parameters to be analyzed include transepithelial electrical resistance, interaction of EhCPADH112 with epithelial surface receptors, changes in expression and localization of epithelial junctional markers and localization of parasite molecules within epithelial cells.

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Human infection by Entamoeba histolytica leads to amoebiasis, a major cause of diarrhea in tropical countries. Infection is initiated by pathogen interactions with intestinal epithelial cells, provoking the opening of cell-cell contacts and consequently diarrhea, sometimes followed by liver infection. This article provides a model to assess the early host-pathogen interactions to improve our understanding of amoebiasis pathogenesis.

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is ...

Simplified Human Neutrophil Extracellular Traps (NETs) Isolation and Handling

Neutrophil Extracellular Traps (NETs) have been recently identified as part of the neutrophil&#8217;s antimicrobial armamentarium. Apart from their role in fighting infections, recent research has demonstrated that they may be involved in many other disease processes, including cancer progression. Isolating purified NETs is a crucial element to allow the study of these functions.
In this video, we demonstrate a simplified method of cell free NET isolation from human whole blood using readily available reagents. Isolated NETs can then be used for immunofluorescence staining, blotting or various functional assays. This enables an assessment of their biologic properties in the absence of the potential confounding effects of neutrophils themselves.
A density gradient separation technique is employed to isolate neutrophils from healthy donor whole blood. Isolated neutrophils are then stimulated by phorbol 12-myristate 13-acetate (PMA) to induce NETosis. Activated neutrophils are then discarded, and a cell-free NET stock is obtained.
We then demonstrate how isolated NETs can be used in an adhesion assay with A549 human lung cancer cells. The NET stock is used to coat the wells of a 96 well cell culture plate O/N, and after ensuring an adequate NET monolayer formation on the bottom of the wells, CFSE labeled A549 cells are added. Adherent cells are quantified using a Nikon TE300 fluorescent microscope. In some wells, 1000U DNAse1 is added 10 min before counting to degrade NETs

Simplified Human Neutrophil Extracellular Traps NETs Isolation and Handling

In the following protocol, we describe a very simple way to isolate Neutrophil Extracellular traps (NETs) from human whole blood using readily available reagents. We then demonstrate how the isolated NETs can be used in an in vitro adhesion assay with cancer cells.

Neutrophil Extracellular Traps (NETs) have been recently identified as part of the neutrophil&#8217;s antimicrobial armamentarium. Apart from their role ...

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 ...

Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

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

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

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

Characterization of Human Monocyte-derived Dendritic Cells by Imaging Flow Cytometry: A Comparison between Two Monocyte Isolation Protocols

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and pathogenesis of inflammation. Isolation and study of DCs have been important in biological research because of their distinctive features. Although they are essential key mediators of the immune system, DCs are very rare in blood, accounting for approximately 0.1 - 1% of total blood mononuclear cells. Therefore, alternatives for isolation methods rely on the differentiation of DCs from monocytes isolated from peripheral blood mononuclear cells (PBMCs). The utilization of proper isolation techniques that combine simplicity, affordability, high purity, and high yield of cells is imperative to consider. In the current study, two distinct methods for the generation of DCs will be compared. Monocytes were selected by adherence or negatively enriched using magnetic separation procedure followed by differentiation into DCs with IL-4 and GM-CSF. Monocyte and MDDC viability, proliferation, and phenotype were assessed using viability dyes, MTT assay, and CD11c/ CD14 surface marker analysis by imaging flow cytometry. Although the magnetic separation method yielded a significant higher percentage of monocytes with higher proliferative capacity when compared to the adhesion method, the findings have demonstrated the ability of both techniques to simultaneously generate monocytes that are capable of proliferating and differentiating into viable CD11c+ MDDCs after seven days in culture. Both methods yielded &gt; 70% CD11c+ MDDCs. Therefore, our results provide insights that contribute to the development of reliable methods for isolation and characterization of human DCs.

This study compares two different methods of human monocyte isolation for obtaining in vitro dendritic cells (DCs). Monocytes are selected by adherence or negatively enriched by magnetic separation. Monocyte yield and viability along with MDDC viability, proliferation and CD11c/CD14 surface marker expression will be compared between both methods.

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and ...

Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal disease. Macrophages are heterogeneous cells that have been implicated in kidney injury. Macrophages may be classified into two different phenotypes: classically activated macrophages (M1 macrophages), that release pro-inflammatory cytokines and promote fibrosis; and alternatively activated macrophages (M2 macrophages) that are associated with immunoregulatory and tissue-remodeling functions. These macrophage phenotypes need to be discriminated and analyzed to determine their contribution to renal injury. However, there are scarce studies reporting consistent phenotypic and functional information about macrophage subtypes in inflammatory renal disease models, especially in rats. This fact may be related to the limited macrophage markers used in rats, contrary to mice. Therefore, novel strategies are necessary to quantify and characterize the renal content of these infiltrating cells in a reliable way. This manuscript details a protocol for kidney digestion and further phenotypic and quantitative analysis of macrophages from rat kidneys by flow cytometry. Briefly, kidneys were incubated with collagenase and total macrophages were identified according to the dual presence of CD45 (leukocytes common antigen) and CD68 (PAN macrophage marker) in live cells.This was followed by surface staining of CD86 (M1 marker) and CD163 (M2 marker). Rat peritoneal macrophages were used as positive control for macrophage marker detection by flow cytometry. Our protocol resulted in low cellular mortality and allowed characterization of different intracellular and surface protein markers, thus limiting the loss of cellular integrity observed in other protocols. Moreover, this procedure allows the use of macrophages for further techniques, including cell sorting and mRNA or protein expression studies, among others.

This manuscript describes a detailed protocol for phenotypic and quantitative analysis of resident macrophages from rat kidneys by flow cytometry. The resulting stained cells can be also used for other applications, including cell sorting, gene expression analysis or functional studies, thus increasing the information obtained in the experimental model.

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal ...

Flow Virometry to Analyze Antigenic Spectra of Virions and Extracellular Vesicles

Cells release small extracellular vesicles (EVs) into the surrounding media. Upon virus infection cells also release virions that have the same size of some of the EVs. Both virions and EVs carry proteins of the cells that generated them and are antigenically heterogeneous. In spite of their diversity, both viruses and EVs were characterized predominantly by bulk analysis. Here, we describe an original nanotechnology-based high throughput method that allows the characterization of antigens on individual small particles using regular flow cytometers. Viruses or extracellular vesicles were immunocaptured with 15 nm magnetic nanoparticles (MNPs) coupled to antibodies recognizing one of the surface antigens. The captured virions or vesicles were incubated with fluorescent antibodies against other surface antigens. The resultant complexes were separated on magnetic columns from unbound antibodies and analyzed with conventional flow cytometers triggered on fluorescence. This method has wide applications and can be used to characterize the antigenic composition of any viral- and non-viral small particles generated by cells in vivo and in vitro. Here, we provide examples of the usage of this method to evaluate the distribution of host cell markers on individual HIV-1 particles, to study the maturation of individual Dengue virions (DENV), and to investigate extracellular vesicles released into the bloodstream.

Viruses or extracellular vesicles were immunocaptured with 15 nm magnetic nanoparticles coupled to antibodies recognizing surface antigens. The captured virions or vesicles were labeled with fluorescent antibodies against other surface antigens. The resultant complexes were separated in high magnetic field and analyzed with conventional flow cytometers triggered on fluorescence.

Cells release small extracellular vesicles (EVs) into the surrounding media. Upon virus infection cells also release virions that have the same size of ...

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. Diet-induced obesity (DIO) is a valuable model in studying the role of macrophage heterogeneity; however, adequate macrophage isolations are difficult to acquire from inflamed tissues. In this protocol, we outline the isolation steps and necessary troubleshooting guidelines derived from our studies for obtaining a suitable population of tissue-resident macrophages from mice following 18 weeks of high-fat (HFD) or high-fat/high-cholesterol (HFHCD) diet intervention. This protocol focuses on three hallmark tissues studied in obesity and atherosclerosis including the liver, white adipose tissues (WAT), and the aorta. We highlight how dualistic usage of flow cytometry can achieve a new dimension of isolation and characterization of tissue-resident macrophages. A fundamental section of this protocol addresses the intricacies underlying tissue-specific enzymatic digestions and macrophage isolation, and subsequent cell-surface antibody staining for flow cytometric analysis. This protocol addresses existing complexities underlying fluorescent-activated cell sorting (FACS) and presents clarifications to these complexities so as to obtain broad range characterization from adequately sorted cell populations. Alternate enrichment methods are included for sorting cells, such as the dense liver, allowing for flexibility and time management when working with FACS. In brief, this protocol aids the researcher to evaluate macrophage heterogeneity from a multitude of inflamed tissues in a given study and provides insightful troubleshooting tips that have been successful for favorable cellular isolation and characterization of immune cells in DIO-mediated inflammation.

This protocol allows a researcher to isolate and characterize tissue-resident macrophages in various hallmark inflamed tissues extracted from diet-induced models of metabolic disorders.

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. ...

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 and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

Extracellular vesicles (EVs) are membranous vesicles released by a variety of cells into the extracellular microenvironment. EVs represent a population of heterogeneous vesicles, whose size range between 40 and 1,000 nm. Accumulated evidence indicated that EVs play important regulatory roles in pathogen-host interactions. A deep understanding of schistosome EVs should provide insights into the mechanisms underlying schistosome-host interactions, enabling development of novel strategies against schistosomiasis. Here, we aim to further study EVs functions in schistosomes by presenting a protocol for the isolation and characterization of EVs from adult Schistosoma japonicum (S. japonicum). EVs were isolated from in vitro culture medium using centrifugation combined with a commercial exosome isolation kit. The isolated S. japonicum EVs (SjEVs) typically possess a diameter of 100 - 400 nm, and are characterized by transmission electronic microscopy and western blotting. The usage of PKH67 dye-labeled SjEVs has demonstrated that SjEVs are internalized by the recipient cells. Overall, our protocol provides an alternative method for isolating EVs from adult schistosomes; the isolated SjEVs may be suitable for functional analysis.

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

Here, we present a protocol to describe extracellular vesicles (EVs) isolation in in vitro cultured medium from adult Schistosoma japonicum.

Extracellular vesicles (EVs) are membranous vesicles released by a variety of cells into the extracellular microenvironment. EVs represent a population of ...