Name: isolation of low endotoxin content extracellular vesicles derived from cancer cell lines
Uploaded: 2023-02-17T13:30:00
Description: Watch this Scientific Journal Video about Isolation of Low Endotoxin Content Extracellular Vesicles Derived from Cancer Cell Lines at JoVE.com

This work was supported by the National Science Centre, Poland, grant number 2019/33/B/NZ5/00647. We would like to thank Professor Tomasz Gosiewski and Agnieszka Krawczyk from the Department of Molecular Medical Microbiology, Jagiellonian University Medical College for their invaluable help in the detection of bacterial DNA in EVs.

1. Preparation of ultracentrifuge tubes
<ol>
	<li>Use sterile, single-use tubes. If this is not possible, reuse the ultracentrifuge tubes after washing them with a detergent using a sterile Pasteur pipette or other single-use applicators. Remember that ultracentrifuge tubes should be dedicated to one type of centrifuged material (cell culture supernatant/serum/plasma) and species (human/mouse/etc.).</li>
	<li>After a detergent wash, rinse the ultracentrifuge tubes with deionized, LPS-free water 3x.<br ...

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LPS+induced+inflammatory+responses+in+human+peripheral+blood+mononuclear+cells+is+mediated+through+NOX4+and+Gi&#945;+dependent+PI-3+kinase+signalling.">LPS induced inflammatory responses in human peripheral blood mononuclear cells is mediated through NOX4 and Gi&#945; dependent PI-3 kinase signalling.</a> Journal of Inflammation. 9 (1), (2012).</li><li>Schwarz, H., Schmittner, M., Duschl, A., Horejs-Hoeck, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Residual+endotoxin+contaminations+in+recombinant+proteins+are+sufficient+to+activate+human+CD1c++dendritic+cells.">Residual endotoxin contaminations in recombinant proteins are sufficient to activate human CD1c+ dendritic cells.</a> PLOS ONE. 9 (12), 113840 (2014).</li><li>Zanoni, I., Granucci, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+CD14+in+host+protection+against+infections+and+in+metabolism+regulation.">Role of CD14 in host protection against infections and in metabolism regulation.</a> Frontiers in Cellular and Infection Microbiology. 3, 32 (2013).</li><li>Takashiba, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+monocytes+to+macrophages+primes+cells+for+lipopolysaccharide+stimulation+via+accumulation+of+cytoplasmic+nuclear+factor+kB.">Differentiation of monocytes to macrophages primes cells for lipopolysaccharide stimulation via accumulation of cytoplasmic nuclear factor kB.</a> Infection and Immunity. 67 (11), 5573-5578 (1999).</li><li>Schwarz, H., 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=Biological+activity+of+masked+endotoxin.">Biological activity of masked endotoxin.</a> Scientific Reports. 7, 44750 (2017).</li><li>Danesh, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Granulocyte-derived+extracellular+vesicles+activate+monocytes+and+are+associated+with+mortality+in+intensive+care+unit+patients.">Granulocyte-derived extracellular vesicles activate monocytes and are associated with mortality in intensive care unit patients.</a> Frontiers in Immunology. 9, 956 (2018).</li><li>Barry, O. 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In the last few years, methods for proper EVs isolation have become increasingly important, enabling their further reliable analyses, for example, in the context of obtaining reliable omics and functional data24. Based on previous research experience, it seems that not only the type of isolation method, but also other conditions during this procedure may be important. The use of EV-depleted FBS is widely recognized as a necessity25,26; how...

Extracellular vesicles (EVs), according to the MISEV 2018 nomenclature, are a collective term describing various subtypes of cell-secreted membranous vesicles that play crucial roles in numerous physiological and pathological processes1,2. Moreover, EVs show promise as novel biomarkers for various diseases, as well as therapeutic agents and drug delivery vehicles. However, realizing their full potential is difficult as there are still many technical obstacles related to their acquisition3. One such challenge is the isolation of endotoxin-free EVs, which has been neglected in many cases. One of the most common endotoxins is lipopolysaccharide (LPS), which is a major component of gram-negative bacterial cell walls and can cause an acute inflammatory response, owing to the release of a large number of inflammatory cytokines by various cells4,5. LPS induces a response by binding to LPS binding protein, followed by interaction with the CD14/TLR4/MD2 complex on myeloid cells. This interaction leads to the activation of MyD88- and TRIF-dependent signaling pathways, which in turn triggers the nuclear factor kappa B (NFkB). Translocation of NFkB to the nucleus initiates the production of cytokines6. Monocytes and macrophages are highly sensitive to LPS, and their exposure to LPS results in a release of inflammatory cytokines and chemokines (e.g., IL-6, IL-12, CXCL8, and TNF-&#945;)7,8. The CD14 structure enables the binding of different LPS species with similar affinity and serves as a co-receptor for other toll-like receptors (TLRs) (TLR1, 2, 3, 4, 6, 7, and 9)6. The number of studies being conducted on the effects of EVs on monocytes/macrophages is still increasing9,10,11. Especially from the perspective of studying the functions of monocytes, their subpopulations, and other immune cells, the presence of endotoxin and even their masked presence in EVs is of great importance12. The overlooked contamination of EVs with endotoxins may lead to misleading conclusions and hide their true biological activity. In other words, working with monocytic cells requires confidence in the absence of endotoxin contamination13. Potential sources of endotoxins can be water, commercially obtained media and sera, media components and additives, laboratory glassware, and plasticware5,14,15.
Therefore, this study aimed to develop a protocol for the isolation of low endotoxin-containing EVs. The protocol provides simple hints on how to avoid endotoxin contamination during EVs isolation instead of removing endotoxins from EVs. Previously, many protocols have been presented on how to remove endotoxins from, for example, engineered nanoparticles used in nanomedicine; however, none of them are useful for biological structures such as EVs. The effective depyrogenation of nanoparticles can be carried out by ethanol or acetic acid rinsing, heating at 175 &#176;C for 3 h, &#947; irradiation, or triton X-100 treatment; however, these procedures lead to the destruction of EVs16,17.
The presented protocol is a pioneering study focused on avoiding endotoxin impurities in EVs, unlike previous studies on the effect of EVs on monocytes9. Applying proposed principles to laboratory practice may help to obtain reliable research results, which can be crucial when considering the potential use of EVs as therapeutic agents in the clinic12.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#160;Alix (3A9) Mouse mAb&#160;</td>
			<td>Cell Signaling Technology</td>
			<td>2171</td>
			<td></td>
		</tr>
		<tr>
			<td>1250ul Filter Universal Pipette Tips, Clear, Polypropylene, Non-Pyrogenic</td>
			<td>GoogLab Scientific</td>
			<td>GBFT1250-R-NS</td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACSCanto II Flow Cytometr</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CBA Human Th1/Th2 Cytokine Kit II</td>
			<td>BD Biosciences</td>
			<td>551809</td>
			<td></td>
		</tr>
		<tr>
			<td>CD9 (D8O1A) Rabbit mAb</td>
			<td>Cell Signaling Technology</td>
			<td>13174</td>
			<td></td>
		</tr>
		<tr>
			<td>ChemiDoc Imaging System</td>
			<td>Bio-Rad Laboratories, Inc.&#160;</td>
			<td>17001401</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM (Dulbecco&#8217;s Modified Eagle&#8217;s Medium)&#160;</td>
			<td>Corning</td>
			<td>10-013-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>ELX800NB, Universal Microplate Reader</td>
			<td>BIO-TEK INSTRUMENTS, INC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>16000044</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum South America Ultra Low Endotoxin</td>
			<td>&#160;Biowest</td>
			<td>&#160;S1860-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin, 50 mg/mL</td>
			<td>&#160;PAN &#8211; Biotech</td>
			<td>P06-13100</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Mouse IgG- HRP</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-2004</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG- HRP</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-2005</td>
			<td></td>
		</tr>
		<tr>
			<td>Immun-Blot PVDF Membrane</td>
			<td>Bio-Rad Laboratories, Inc.&#160;</td>
			<td>1620177</td>
			<td></td>
		</tr>
		<tr>
			<td>LPS from Salmonella abortus equi S-form (TLRGRADE)</td>
			<td>&#160;Enzo Life Sciences, Inc.</td>
			<td>ALX-581-009-L002</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini Trans-Blot Electrophoretic Transfer Cell</td>
			<td>Bio-Rad Laboratories, Inc.&#160;</td>
			<td>1703930</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanoparticle Tracking Analysis&#160;</td>
			<td>Malvern Instruments Ltd</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NuPAGE LDS Sample Buffer (4X)</td>
			<td>&#160;Invitrogen</td>
			<td>&#160;NP0007</td>
			<td></td>
		</tr>
		<tr>
			<td>NuPAGE Sample Reducing Agent (10x)</td>
			<td>&#160;Invitrogen</td>
			<td>NP0004</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Sigma Aldrich</td>
			<td>P7793</td>
			<td>transparent film</td>
		</tr>
		<tr>
			<td>Perfect 100-1000 bp DNA Ladder</td>
			<td>EURx</td>
			<td>E3141-01&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>PierceTM Chromogenic Endotoxin Quant Kit</td>
			<td>Thermo Scientific</td>
			<td>A39552</td>
			<td></td>
		</tr>
		<tr>
			<td>PP Oak Ridge Tube with sealing caps</td>
			<td>Thermo Scientific</td>
			<td>3929, 03613</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>RPMI-1640 (Gibco)</td>
			<td>11875093</td>
			<td></td>
		</tr>
		<tr>
			<td>SimpliAmp Thermal Cycler</td>
			<td>Applied Biosystem</td>
			<td>A24811</td>
			<td></td>
		</tr>
		<tr>
			<td>Sorvall wX+ ULTRA SERIES Centrifuge with T-1270 rotor</td>
			<td>Thermo Scientific</td>
			<td>75000100</td>
			<td></td>
		</tr>
		<tr>
			<td>Sub-Cell GT Horizontal Electrophoresis System</td>
			<td>Bio-Rad Laboratories, Inc.&#160;</td>
			<td>1704401</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperSignal West Pico PLUS Chemiluminescent Substrate</td>
			<td>Thermo Scientific</td>
			<td>34577</td>
			<td></td>
		</tr>
		<tr>
			<td>SW480 cell line</td>
			<td>American Type Culture Collection(ATCC)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SW480 cell line</td>
			<td>American Type Culture Collection (ATCC)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe filter 0.22 um</td>
			<td>TPP</td>
			<td>99722</td>
			<td></td>
		</tr>
		<tr>
			<td>Trans-Blot SD Semi-Dry Transfer Cell</td>
			<td>Bio-Rad Laboratories, Inc.&#160;</td>
			<td>1703940</td>
			<td>Transfer machine</td>
		</tr>
		<tr>
			<td>Transfer pipette, 3.5 mL</td>
			<td>SARSTEDT</td>
			<td>86.1171.001</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of low endotoxin content extracellular vesicles derived from cancer cell lines

Extracellular vesicles (EVs) are a heterogeneous population of membrane vesicles released by cells in vitro and in vivo. Their omnipresence and significant role as carriers of biological information make them intriguing study objects, requiring reliable and repetitive protocols for their isolation. However, realizing their full potential is difficult as there are still many technical obstacles related to their research (like proper acquisition). This study presents a protocol for the isolation of small EVs (according to the MISEV 2018 nomenclature) from the culture supernatant of tumor cell lines based on differential centrifugation. The protocol includes guidelines on how to avoid contamination with endotoxins during the isolation of EVs and how to properly evaluate them. Endotoxin contamination of EVs can significantly hinder subsequent experiments or even mask their true biological effects. On the other hand, the overlooked presence of endotoxins may lead to incorrect conclusions. This is of particular importance when referring to cells of the immune system, including monocytes, because monocytes constitute a population that is especially sensitive to endotoxin residues. Therefore, it is highly recommended to screen EVs for endotoxin contamination, especially when working with endotoxin-sensitive cells such as monocytes, macrophages, myeloid-derived suppressor cells, or dendritic cells.

A prerequisite or obligatory step for this protocol is the exclusion of possible endotoxin contamination from reagents. All the reagents being used, such as FBS, DMEM, RPMI, PBS, and even ultracentrifuge tubes, must be endotoxin-free (&#60;0.005 EU/mL). Maintaining the regime of no endotoxin contamination is not easy as, for example, the regular/standard serum for cell culture can be its rich source (0.364 EU/mL; see Table 1).
Although this protocol was developed to isolate EV...

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Isolation of Low Endotoxin Content Extracellular Vesicles Derived from Cancer Cell Lines

The proposed protocol includes guidelines on how to avoid contamination with endotoxin during the isolation of extracellular vesicles from cell culture supernatants, and how to properly evaluate them.

Extracellular vesicles (EVs) are a heterogeneous population of membrane vesicles released by cells in vitro and in vivo. Their omnipresence and ...

This protocol provide guidelines on how to avoid contamination with endotoxin during the isolation of extracellular vesicles from cell culture supernatants, and how to properly evaluate them. The main advantage of this protocol is that every lab concerned with EVs may limit endotoxin contamination by keeping an aseptic procedure and using simple and widely available equipment. Everyone who is trying this technique for the first time should start with new media, reagents, and plasticware which are low-endotoxin, non-pyrogenic labeled.For this protocol, use endotoxin-free reagents, water, culture media, filtered PBS, ultra low-endotoxin FBS, and ultracentrifuge tubes. To begin, collect the supernatant from previously cultured SW480 and SW620 cell lines into appropriately labeled 15-milliliter tubes. Remove cell debris by centrifuging the supernatant at 500 x g for five minutes at room temperature.Without disturbing the debris, collect the supernatant into a new labeled tube, and centrifuge at 3, 200 x g for 12 minutes at four degrees Celsius. Carefully transfer 45 milliliters of the supernatant into a vertically-placed sterile 50-milliliter tube, without wetting the edges of the tube. Wrap the tube's cap with a sterile, transparent film, and store it vertically at minus 80 degrees Celsius for subsequent isolation of extracellular vesicles.Begin isolation by preparing 0.22-micrometer syringe filters, syringes, and tubes containing approximately 90 milliliters of supernatant. Fill a syringe with the supernatant, and fix the filter to the needle adapter. Filter the supernatant through the filter into a 50-milliliter tube.Pipette seven milliliters of the filtrate into a prepared ultracentrifuge tube, and centrifuge at 100, 000 x g for two hours at four degrees Celsius. Use a sterile Pasteur pipette to discard the supernatant. Pull the pellets into an ultracentrifuge tube using a long filtered pipette tip.Rinse the extracellular vesicles by adding seven milliliters of filtered endotoxin-free PBS. After ultracentrifugation, discard the supernatant using a sterile Pasteur pipette and resuspend the pellet in 200 microliters of endotoxin-free PBS. Transfer the suspension to a sterile 1.5-milliliter low protein binding test tube.Then transfer 10 microliters of suspension to a new 1.5-milliliter tube for further analysis. Wrap the cap of the tube, and store it at minus 80 degrees Celsius. Now, into a new tube, dilute one microliter of suspension using filtered PBS at a ratio of 1 to 1000 for nanoparticle tracking analysis.To perform blotting analysis, first mix 20 micrograms of the samples with a loading buffer. Incubate the samples at 70 degrees Celsius for 10 minutes. Then load 20 micrograms of the sample into each well of a 10 to 14%polyacrylamide gel with SDS, and perform electrophoresis for 45 minutes at 150 volts, with running buffer.Next, place the gel in a transfer machine with Towbin buffer, and perform a semi-dry transfer of proteins onto a polyvinylidene difluoride membrane at 25 volts for one hour. Block the membrane with 1%BSA by incubating it for one hour on a rocker. Add BSA diluted antibodies, anti-CD9 or anti-Alix, to the membrane, and incubate overnight at four degrees Celsius on a rocker.After removing the antibodies, wash the membrane thrice with 10 milliliters of TBST for 10 minutes. Now add goat anti-rabbit or anti-mouse secondary antibody, and incubate again on a rocker for one hour at room temperature. Discard the antibody, and wash the membrane as demonstrated previously.Pour one milliliter of the solution containing substrate and luminol at an equal ratio on the membrane. Immediately place the membrane in an imaging system, and visualize the protein bands on the screen. In the present study, western blot analysis confirmed the presence of two extracellular vesicle markers, CD9 and Alix.The mean size and concentrations of the vesicles isolated from both SW480 and SW620 were similar. Stimulation of monocyte with different doses of lipopolysaccharide, or LPS, showed the lowest dose of LPS enabling the secretion of interleukin 10 and tumor necrosis factor in monocytes was 50 picogram per milliliter. The chromogenic LAL test indicated that LPS contamination of the extracellular vesicles was around 50 picograms per milliliter for both cell lines.The most important thing to remember is the use of LPS-free or depleted reagents, and routine LPS measurement at each step of the procedure. Preventing endotoxin contamination is easier than eliminating it. The proposed protocol limits the possibility of false results due to the contamination of EVs with endotoxin, and allows for assessing the EVs activity per cell.The proposed protocol is useful for researchers working with endotoxin-sensitive cells, like monocytes, dendritic cells, and macrophages.

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Research

JoVE Journal

Immunology and Infection

Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice

Established cell lines are a critical research tool that can reduce the use of laboratory animals in research. Certain strains of genetically modified mice, such as Atm-/- and p53-/- consistently develop thymic lymphoma early in life 1,2, and thus, can serve as a reliable source for derivation of murine T-cell lines. Here we present a detailed protocol for the development of established murine thymic lymphoma T-cell lines without the need to add interleukins as described in previous protocols 1,3. Tumors were harvested from mice aged three to six months, at the earliest indication of visible tumors based on the observation of hunched posture, labored breathing, poor grooming and wasting in a susceptible strain 1,4. We have successfully established several T-cell lines using this protocol and inbred strains ofAtm-/- [FVB/N-Atmtm1Led/J] 2 and p53-/- [129/S6-Trp53tm1Tyj/J] 5 mice. We further demonstrate that more than 90% of the established T-cell population expresses CD3, CD4 and CD8. Consistent with stably established cell lines, the T-cells generated by using the present protocol have been passaged for over a year.

Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice

In this video we demonstrate a protocol to establish mouse thymic lymphoma cell lines. By following this protocol, we have successfully established several T-cell lines from Atm-/- and p53-/- mice with thymic lymphoma.

Established cell lines are a critical research tool that can reduce the use of laboratory animals in research. Certain strains of genetically modified ...

Establishment of Epstein-Barr Virus Growth-transformed Lymphoblastoid Cell Lines

Infection of B cells with Epstein-Barr virus (EBV) leads to proliferation and subsequent immortalization, resulting in establishment of lymphoblastoid cell lines (LCL) in vitro. Since LCL are latently infected with EBV, they provide a model system to investigate EBV latency and virus-driven B cell proliferation and tumorigenesis1. LCL have been used to present antigens in a variety of immunologic assays2, 3. In addition, LCL can be used to generate human monoclonal antibodies4, 5 and provide a potentially unlimited source when access to primary biologic materials is limited6, 7.
A variety of methods have been described to generate LCL. Earlier methods have included the use of mitogens such as phytohemagglutinin, lipopolysaccharide8, and pokeweed mitogen9 to increase the efficiency of EBV-mediated immortalization. More recently, others have used immunosuppressive agents such as cyclosporin A to inhibit T cell-mediated killing of infected B cells7, 10-12.
The considerable length of time from EBV infection to establishment of cell lines drives the requirement for quicker and more reliable methods for EBV-driven B cell growth transformation. Using a combination of high titer EBV and an immunosuppressive agent, we are able to consistently infect, transform, and generate LCL from B cells in peripheral blood. This method uses a small amount of peripheral blood mononuclear cells that are infected in vitroclusters of cells can be demonstrated. The presence of CD23 with EBV in the presence of FK506, a T cell immunosuppressant. Traditionally, outgrowth of proliferating B cells is monitored by visualization of microscopic clusters of cells about a week after infection with EBV. Clumps of LCL can be seen by the naked eye after several weeks. We describe an assay to determine early if EBV-mediated growth transformation is successful even before microscopic clusters of cells can be demonstrated. The presence of CD23hiCD58+ cells observed as early as three days post-infection indicates a successful outcome.

We describe a method for generating transformed B cell lines using Epstein-Barr virus. We also illustrate a novel assay that can identify B cells destined to undergo transformation as early as three days after infection.

Infection of B cells with Epstein-Barr virus (EBV) leads to proliferation and subsequent immortalization, resulting in establishment of lymphoblastoid ...

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for isolating precursor B-cells at four different stages of differentiation. Because genes are expressed and epigenetic modifications occur in a tissue specific manner, it is vital to discriminate between tissues and cell types in order to be able to identify alterations in the genome and the epigenome that may lead to the development of disease. This method can be adapted to any type of cell present in umbilical cord blood at any stage of differentiation.	
This method comprises 4 main steps. First, mononuclear cells are separated by density centrifugation. Second, B-cells are enriched using biotin conjugated antibodies that recognize and remove non B-cells from the mononuclear cells. Third the B-cells are fluorescently labeled with cell surface protein antibodies specific to individual stages of B-cell development. Finally, the fluorescently labeled cells are sorted and individual populations are recovered. The recovered cells are of sufficient quantity and quality to be utilized in downstream nucleic acid assays.

Here we describe a protocol for isolating subsets of precursor B-cells from umbilical cord blood. A sufficient quantity and quality of nucleic acids may be extracted from the cells and used in subsequent assays utilizing DNA or RNA.

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

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 ...

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 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 ...

Isolation of Extracellular Vesicles from Murine Bronchoalveolar Lavage Fluid Using an Ultrafiltration Centrifugation Technique

Extracellular vesicles (EVs) are newly discovered subcellular components that play important roles in many biological signaling functions during physiological and pathological states. The isolation of EVs continues to be a major challenge in this field, due to limitations intrinsic to each technique. The differential ultracentrifugation with density gradient centrifugation method is a commonly used approach and is considered to be the gold standard procedure for EV isolation. However, this procedure is time-consuming, labor-intensive, and generally results in low scalability, which may not be suitable for small-volume samples such as bronchoalveolar lavage fluid. We demonstrate that an ultrafiltration centrifugation isolation method is simple and time- and labor-efficient yet provides a high recovery yield and purity. We propose that this isolation method could be an alternative approach that is suitable for EV isolation, particularly for small-volume biological specimens.

Here, we describe two extracellular vesicle isolation protocols, ultrafiltration centrifugation and ultracentrifugation with density gradient centrifugation, to isolate extracellular vesicles from murine bronchoalveolar lavage fluid samples. The extracellular vesicles derived from murine bronchoalveolar lavage fluid by both methods are quantified and characterized.

Extracellular vesicles (EVs) are newly discovered subcellular components that play important roles in many biological signaling functions during ...

Purification of High Yield Extracellular Vesicle Preparations Away from Virus

One of the major hurdles in the field of extracellular vesicle (EV) research today is the ability to achieve purified EV preparations in a viral infection setting. The presented method is meant to isolate EVs away from virions (i.e., HIV-1), allowing for a higher efficiency and yield compared to conventional ultracentrifugation methods. Our protocol contains three steps: EV precipitation, density gradient separation, and particle capture. Downstream assays (i.e., Western blot, and PCR) can be run directly following particle capture. This method is advantageous over other isolation methods (i.e., ultracentrifugation) as it allows for the use of minimal starting volumes. Furthermore, it is more user friendly than alternative EV isolation methods requiring multiple ultracentrifugation steps. However, the presented method is limited in its scope of functional EV assays as it is difficult to elute intact EVs from our particles. Furthermore, this method is tailored towards a strictly research-based setting and would not be commercially viable.

This protocol isolates extracellular vesicles (EVs) away from virions with high efficiency and yield by incorporating EV precipitation, density gradient ultracentrifugation, and particle capture to allow for a streamlined workflow and a reduction of starting volume requirements, resulting in reproducible preparations for use in all EV research.

One of the major hurdles in the field of extracellular vesicle (EV) research today is the ability to achieve purified EV preparations in a viral infection ...

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.

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) ...