We thank Prof. Dr. Markus Schnare for providing us with TLR2-/- and TLR2/4-/- mice and Prof. Dr. Carsten Kirschning for TRIF/MyD88-/- mice. Parts of this work was funded by Bundesministerium f&#252;r Bildung und Forschung (e:bio miRSys - FKZ 0316175B, e:Med CAPSYS - FKZ 01X1304E; http://www.bmbf.de/), Deutsche Forschungsgemeinschaft (SFB/TR-84; http://www.sfb-tr84.de/), and Hessisches Ministerium f&#252;r Wissenschaft und Kunst (LOEWE Medical RNomics - FKZ 519/03/00.001-(0003); http://www.proloewe.de/medicalrnomics), all to BS.

1. Prepare Medium and Agar Plates
<ol>
	<li>Prepare 1 L of broth medium (YEB). Dissolve 10 g of ACES and 10 g of yeast extract in 900 mL of distilled water. Adjust the pH to 6.9 with KOH (5 N). Add 10 mL of L-cysteine (0.4 g in 10 mL of distilled water) and 10 mL of Fe(NO3)3x9H2O (0.25 g in 10 mL of distilled water). Fill up to 1 L with distilled water and filter sterilize the solution (pore size: 0.22 &#181;m). Store at 4 &#176;C.</li>
	<li>Prepare buffered charcoal yeast extract (BCYE) agar plates. Dissolve 10 g of ACES and 10 g of yeast extract in 900 mL of distilled water. Adjust the pH to 6.9 with KOH (5 N). Add 15 g of agar and 2.5 g of activated charcoal. Fill up to 1 L with distilled water and autoclave.
	<ol>
		<li>Add 10 mL of L-cysteine (0.4 g in 10 mL of distilled water) and 10 mL of Fe(NO3)3x9H2O (0.25 g in 10 mL of distilled water, both sterilized by filtration through 0.22-&#181;m pores) to cooled BCYE (approximately 50 &#176;C). Pour plates and store at 4 &#176;C.</li>
	</ol>
	</li>
</ol>
2. Cultivate L. pneumophila
<ol>
	<li>Spread L. pneumophila strain Corby (wild type, WT) on BCYE agar plates and incubate them at 37 &#176;C for 3 days. Inoculate 10 mL of YEB at an OD600 of 0.3 with L. pneumophila from the preculture plate; incubate the bacteria at 37 &#176;C on a rotating shaker (150 rpm) for 6 h.</li>
	<li>Verify the purity of the liquid culture by spreading 100 &#181;L of the suspension on a blood agar plate. Incubate overnight at 37 &#176;C.</li>
	<li>Add the remaining liquid culture to 90 mL of fresh YEB medium and incubate on a rotating shaker (37 &#176;C and 150 rpm) to reach an OD600 of 3.0-3.5, which takes approximately 16-20 h.</li>
</ol>
3. Prepare and Quantify L. pneumophila OMVs
NOTE: Carry out all of the following centrifugation steps under sterile conditions and at 4 &#176;C.
<ol>
	<li>Centrifuge the liquid culture at 4,000 x g for 20 min to pellet the bacteria. Transfer the supernatant to fresh centrifuge tubes, discard the bacterial pellet, and repeat the centrifugation (4,000 x g for 20 min). Repeat this step once.</li>
	<li>Sterile-filter the remaining supernatant twice (pore size: 0.22 &#181;m). Transfer the bacteria-free supernatant to ultracentrifuge tubes and ultracentrifuge at 100,000 x g for 3 h.</li>
	<li>Decant the supernatant and discard it. Resuspend the OMV pellet in sterile phosphate-buffered saline (PBS) and ultracentrifuge (100,000 x g for 3 h) to remove contaminating proteins and LPS.</li>
	<li>Discard the supernatant and resuspend the OMV pellet in 500 &#181;L of sterile PBS. Streak 20 &#181;L on a blood agar plate and on a BCYE agar plate to exclude bacterial contamination of the prepared vesicles. Incubate the blood agar plate overnight and the BCYE agar plate for 3 days (both at 37 &#176;C).</li>
	<li>Quantify the protein amount obtained from the OMV preparation using a bicinchoninic acid assay according to the manufacturer&#39;s instructions. 
	NOTE: The concentration of 100 mL of L. pneumophila culture is usually 1 &#181;g/&#181;L. Store the prepared and quantified OMVs at -20 &#176;C.</li>
</ol>
4. Pre-treat Macrophages
<ol>
	<li>Prepare THP-1 cells. 
	NOTE: THP-1 is a monocytic cell line derived from a leukemia patient.
	<ol>
		<li>Add 2x105 THP-1 cells per 24 wells and differentiate them by adding 20 nM phorbol 12-myristate 13-acetate (PMA) into macrophage-like cells. Incubate for 24 h at 37 &#176;C.</li>
		<li>Replace the medium with 500 &#181;L of fresh medium and incubate for another 24 h; the optimal medium for THP-1 cells is composed of RPMI 1640 high glucose supplemented with 10% fetal calf serum.</li>
	</ol>
	</li>
	<li>Isolate the murine bone marrow-derived macrophages (mBMDM), as described in Reference 20.</li>
	<li>Treat THP-1-derived macrophages or mBMDM with OMVs.
	<ol>
		<li>Thaw the OMVs prepared in step 3 and add them according to their protein amount (0.1, 1, and 10 &#181;g/mL) to the human or murine macrophages. Incubate the macrophages with OMVs at 37 &#176;C for at least 20 h. Use the supernatant for ELISA or move on with step 5.</li>
	</ol>
	</li>
</ol>
5. Infect the Macrophages and Assess Bacterial Replication with a Colony-Forming Unit (CFU) Assay
<ol>
	<li>Use L. pneumophila from step 2.1, pre-treated THP-1 cells or mBMDM from step 4.3, and not pre-treated macrophages as controls (2x105/24 wells). Do not exchange the medium.</li>
	<li>Infect THP-1 cells with L. pneumophila Corby WT and mBMDM with a flagellin-lacking mutant of L. pneumophila Corby (both with a multiplicity of infection (MOI) of 0.5; 1x105 L. pneumophila/24 wells) and incubate for 24 and 48 h, respectively. Prepare both L. pneumophila Corby (WT or flagellin-lacking mutant) as described in step 2.1.</li>
	<li>Lyse the cells in their medium by the addition of saponin (final concentration: 0.1%) and incubate at 37 &#176;C for 5 min.</li>
	<li>Resuspend the bacteria by pipetting and transfer the suspension to a reaction vessel. Prepare serial dilutions of the L. pneumophila-containing media in sterile PBS.</li>
	<li>Streak 50 &#181;L of the required dilutions on BCYE agar plates and incubate for 3 days at 37 &#176;C.</li>
	<li>Visually count the formed colonies. Calculate the CFU <img alt="Equation 1" src="/files/ftp_upload/55146/55146eq1.jpg" />&#160; Normalize the CFU count result to not pre-treated but infected macrophages, which are set to 100%.</li>
</ol>

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Novartis.com Available from: <a target="_blank" href="https://www.novartis.com/news/media-releases/novartis-bexsero%C2%AE-vaccine-approved-fda-prevention-meningitis-b-leading-cause">https://www.novartis.com/news/media-releases/novartis-bexsero%C2%AE-vaccine-approved-fda-prevention-meningitis-b-leading-cause</a> (2016)</li></ol>

The authors have nothing to disclose.

The OMVs of bacterial pathogens and the impact of these membrane vesicles on their target cells are currently being intensively studied. For example, Clostridium perfringens-derived OMVs induce cytokine secretion in macrophages, B lymphocytes can be activated by OMVs from Borrelia burgdorferi, and Helicobacter pylori-released membrane vesicles can act on gastric epithelial cells21,22,23. L. pneumophila, an intracellular pathogen that can induce a severe form of atypical pneumonia, also releases OMVs that are able to activate lung epithelial cells and macrophages16,19. Here, we present a detailed protocol for the small-scale isolation of L. pneumophila OMVs from liquid culture to study the potential role of OMVs in pneumonia. It is critical to work under sterile conditions and to rule out contamination from other bacteria in order to obtain a pure L. pneumophila-derived OMV preparation. The isolation of OMVs includes a filtration step through 0.22-&#181;m pores in order to prevent the contamination of the obtained OMV pellet with L. pneumophila, even though this reduces the OMV yield, since the largest OMVs are lost by this filtration step.
Furthermore, we tested the response of human and murine macrophages to those isolated vesicles and infected cells with L. pneumophila to more closely approximate the situation in Legionella pneumonia, where OMVs are released inside the LCV by extracellular bacteria15. The employed OMV doses have been estimated according to the free OMV amount in an in vitro infection of human macrophages after 24 h of incubation (described in Reference 20). For the stimulation of other recipient cells or in vivo experiments, other OMV doses might be necessary and must be established. The analysis of the effect of L. pneumophila OMVs represents an advancement to the protocol described by Jager and Steinert24.
Here, PMA-differentiated THP-1 cells serve as a model for alveolar macrophages due to the limited availability of primary human material. The addition of PMA differentiates the monocytic THP-1 cells into macrophage-like cells25. Furthermore, they are a well-known model cell line for L. pneumophila studies26. Besides this human monocytic cell line, mBMDM cells are used. mBMDM are widely accepted for the study of the effects of L. pneumophila27,28,29. The possibility of using genetic knockouts for different TLRs or other proteins make them a valuable tool for studying OMV effects. In order to lower the amount of mice per experiment, mBMDM are used instead of alveolar macrophages due to the limitations of the macrophages. Key experiments might require alveolar macrophages for validation.
Besides the herein-described protocol of ultracentrifugation to purify OMVs, it is possible to perform a density gradient centrifugation, which is included in the protocol by Chutkan et al.30. This could improve the purity of the obtained OMV preparation and reduce the amount of co-purified protein aggregates, flagellin, and LPS. The purity of the obtained OMV preparation can be analyzed by transmission electron microscopy or by nanoparticle tracking analysis as a supplementary step in quality control. This can provide an additional means of quantification, beyond the protein measurement procedure presented here. Optionally, the LPS concentration can be analyzed by a limulus amebocyte lysate test. If the OMV yield is low, an additional concentration step via centrifugal filters could be performed, which was not done here. If the yield was lower than expected, the OMVs were discarded.
As part of the ongoing effort to elucidate the biological mechanisms and functions behind OMVs, the influence of different stress conditions on OMV production could be tested. Nutrient deprivation, changes in incubation temperature, or exposure to harmful agents might have an impact on OMV secretion31. Possible stress conditions are discussed in the protocol by Klimentova and Stulik32. Moreover, hyper- or hypovesiculating L. pneumophila mutants could be generated. The different OMV preparations could then be analyzed in infection experiments with macrophages, human lung tissue explants (described in Reference 17), or even in in vivo models. Besides the role of OMVs in innate immune signaling, their influence in bacterial communication can be addressed experimentally. Furthermore, the impact of various innate immune signaling cascades might be analyzed by the use of murine knockout cells or the generation of CRISPR/Cas9 knockouts in human cell lines. This basic research in OMVs will assist in the development of new vaccine strategies, which already exist for meningitis B transmitted by Neisseria meningitides33.
Starting from the protocol on OMV isolation and characterization, one can apply this to other Gram-negative bacteria and to other host cells; it only needs to be adjusted to the growth of the bacteria in liquid culture. The protocol published by Chutkan et al. provides detailed information on the generation of OMVs from Escherichia coli and Pseudomonas aeruginosa30. The culture should not reach the late stationary phase in order to avoid increases in lysed bacteria and contaminating proteins and membranes. Additionally, the OMV dose used for stimulation of the host cells needs to be determined according to the amount of OMVs present during in vivo infections, while still ensuring a low rate of cytotoxicity. In this way, the pathological role of OMVs, their impact on inter-species communication, and host-pathogen interactions could be examined.
To further study the role of L. pneumophila OMVs in pneumonia, standardized OMV preparations with sufficient yields and comparable infection experiments are needed. This protocol will help to standardize isolation procedures and to extend OMV studies to other Gram-negative bacteria and to other host cells. Furthermore, research will benefit from the detailed in vitro knowledge, which can be used to extend experiments to in vivo settings. In the future, this protocol could be extended to the isolation of OMVs from primary biological material, such as serum or bronchoalveolar lavage fluid, to gain insight into the composition of OMVs released under physiological conditions. This will help to determine key parameters of OMV composition and to understand the properties of in vitro-generated OMVs.

Bacteria can secrete virulence factors via different mechanisms1. Besides the well-known secretory systems, Gram-negative bacteria can exchange information and deliver virulence factors via outer membrane vesicles (OMVs), which are small, spheroid vesicles 10-300 nm in diameter and with a bilayered membrane structure. They are secreted in a variety of growth environments (liquid culture, solid culture, and biofilms) and in all growth phases2,3. OMVs are an important means of transportation (e.g., for proteins, adhesins, toxins, and enzymes, as well as for LPS, which is found on the OMV surface)4. The intraluminal cargo is protected from proteolytic degradation, so it is able to act over long distances, and the vesicles can be found in body fluids and distant organs5,6,7,8. They can not only be recognized and taken up by eukaryotic cells9,10, but furthermore, they are able to facilitate the binding of bacteria and their invasion into host cells4. Legionella pneumophila (L. pneumophila) is a Gram-negative bacterium that can release OMVs. In the human lung, it primarily infects alveolar macrophages, even though its natural host are freshwater amoebae11. An L. pneumophila infection can cause Legionnaires&#39; disease, a severe form of pneumonia12. It blocks phagosome-lysosome fusion in the host cell. It also recruits host organelles, whereby a replication niche, the Legionella-containing vacuole (LCV), is formed13,14. Lysosomal degradation is inhibited not only by effector protein translocation via the type IV secretion system, but also by the release of OMVs15.
The purification of OMVs from bacterial cultures is required to analyze their effect on recipient cells. Earlier studies focused on the protein content of L. pneumophila OMVs and on the influence of the vesicles on alveolar epithelial cells16, but later studies with human lung tissue transplants demonstrated that L. pneumophila OMVs are taken up by alveolar macrophages17.
As OMVs present pathogen-associated molecular patterns (PAMPs) and other bacterial antigens, they might have an impact on the infection of eukaryotic cells and modulate the host immune response18. L. pneumophila OMVs rapidly fuse with host cell membranes and, moreover, they activate the membranous TLR219. As it is known that L. pneumophila OMVs stimulate macrophages and epithelial cells in a pro-inflammatory manner16,17, we analyzed the impact of OMVs on the infection process in human and murine macrophages.
Here, we describe a protocol for the cultivation of L. pneumophila in liquid culture to isolate the secreted OMVs by differential ultracentrifugation and to assess the impact of the vesicles on eukaryotic host cells, either directly or following an infection.

<table border="0" cellpadding="0" cellspacing="0" width="1938">
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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">10 cm petridish</td>
			<td style="width:475px;">Sarstedt AG &#38; Co KG (Nuembrecht, Germany)</td>
			<td align="right" style="width:119px;">82.1473</td>
			<td style="width:1128px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">70 Ti rotor</td>
			<td style="width:475px;">Beckman Coulter Incorporation (California, USA)</td>
			<td align="right">337922</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ACES</td>
			<td style="width:475px;">Carl Roth GmbH &#38; Co KG (Karlsruhe, Germany)</td>
			<td align="right">9138.2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">activated charcoal</td>
			<td style="width:475px;">Carl Roth GmbH &#38; Co KG (Karlsruhe, Germany)</td>
			<td style="width:119px;">X865.2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">agar-agar, Kobe I</td>
			<td style="width:475px;">Carl Roth GmbH &#38; Co KG (Karlsruhe, Germany)</td>
			<td align="right">5210.2</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Columbia agar with 5 % sheep blood</td>
			<td style="width:475px;">Becton Dickinson GmbH (Heidelberg, Germany)</td>
			<td align="right" style="width:119px;">254005</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">cuvettes</td>
			<td style="width:475px;">Sarstedt AG &#38; Co KG (Nuembrecht, Germany)</td>
			<td align="right" style="width:119px;">67.742</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">ELISA (human)</td>
			<td style="width:475px;">BD OptEIA&#8482;; Becton Dickinson GmbH (Heidelberg, Germany)</td>
			<td style="width:119px;">IL-8: 555244 IL-6: 550799</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">ELISA (murine)</td>
			<td style="width:475px;">DuoSet, R&#38;D (Minneapolis, USA)</td>
			<td style="width:119px;">CXCL1: DY453-05</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">Fe(NO3)3x9H2O</td>
			<td style="width:475px;">Carl Roth GmbH &#38; Co KG (Karlsruhe, Germany)</td>
			<td align="right">5632.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fetal calf serum (FCS)</td>
			<td>Life Technologies GmbH (Darmstadt, Germany)</td>
			<td>10270-106</td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;">Heracell 240i CO2 incubator</td>
			<td style="width:475px;">Thermo Fisher Scientific Germany BV &#38; Co KG (Braunschweig, Germany)</td>
			<td align="right">40830469</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heraeus Multifuge X3R</td>
			<td>Thermo Fisher Scientific Germany BV &#38; Co KG (Braunschweig, Germany)</td>
			<td align="right">75004515</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Inoculation loop&#160;</td>
			<td style="width:475px;">Sarstedt AG &#38; Co KG (Nuembrecht, Germany)</td>
			<td style="width:119px;">86.1567.010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">KOH</td>
			<td style="width:475px;">Carl Roth GmbH &#38; Co KG (Karlsruhe, Germany)</td>
			<td align="right" style="width:119px;">6751.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">L. pneumophila Corby</td>
			<td style="width:475px;">---</td>
			<td style="width:119px;">---</td>
			<td>kindly provided by Prof Dr Antje Flieger (RKI, Berlin, Germany)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">L. pneumophila Corby &#916;flaA</td>
			<td style="width:475px;">---</td>
			<td style="width:119px;">---</td>
			<td>kindly provided by Prof Dr Klaus Heuner (RKI, Berlin, Germany)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">L-cystein</td>
			<td style="width:475px;">Carl Roth GmbH &#38; Co KG (Karlsruhe, Germany)</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">mBMDM</td>
			<td style="width:475px;">---</td>
			<td style="width:119px;">---</td>
			<td>kindly provided by Prof Dr Markus Schnare (Philipps Univeristy Marburg, Marburg, Germany) and Prof Dr Carsten Kirschning (University Duisburg Essen, Essen, Germany)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PBS</td>
			<td>Biochrom GmbH (Berlin, Germany)</td>
			<td>L 1825</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">phorbol 12-myristate 13-acetate</td>
			<td style="width:475px;">Sigma-Aldrich Chemie GmbH (Taufkirchen, Germany)</td>
			<td>P8139-1MG</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">rotating shaker (MaxQ 6000)</td>
			<td style="width:475px;">Thermo Fisher Scientific Germany BV &#38; Co KG (Braunschweig, Germany)</td>
			<td>SHKE6000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RPMI 1640 high glucose</td>
			<td>Life Technologies GmbH (Darmstadt, Germany)</td>
			<td>11875-093</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">saponin</td>
			<td style="width:475px;">Carl Roth GmbH &#38; Co KG (Karlsruhe, Germany)</td>
			<td align="right" style="width:119px;">9622.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ultrospec 10</td>
			<td style="width:475px;">Biochrom Ltd (Cambridge, England)</td>
			<td style="width:119px;">80-2116-30</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">sterile filter (pore size: 0.22 &#181;m)</td>
			<td style="width:475px;">Corning Incorporated (new York, USA)</td>
			<td align="right" style="width:119px;">431096</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">THP-1</td>
			<td style="width:475px;">Sigma-Aldrich Chemie GmbH (Taufkirchen, Germany)</td>
			<td>88081201-1VL</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sorvall Discovery 100 SE</td>
			<td style="width:475px;">Thermo Fisher Scientific Germany BV &#38; Co KG (Braunschweig, Germany)</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">yeast extract</td>
			<td>Carl Roth GmbH &#38; Co KG (Karlsruhe, Germany)</td>
			<td align="right">2363.2</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Pierce BCA protein assay kit</td>
			<td style="width:475px;">Thermo Fisher Scientific Germany BV &#38; Co KG (Braunschweig, Germany)</td>
			<td align="right" style="width:119px;">23225</td>
			<td></td>
		</tr>
	</tbody>
</table>

legionella pneumophila outer membrane vesicles: isolation and analysis of their pro-inflammatory potential on macrophages

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or directly into another cell, Gram-negative bacteria can also form outer membrane vesicles (OMVs). These membrane vesicles can deliver their cargo over long distances, and the cargo is protected from degradation by proteases and nucleases. 
Legionella pneumophila (L. pneumophila) is an intracellular, Gram-negative pathogen that causes a severe form of pneumonia. In humans, it infects alveolar macrophages, where it blocks lysosomal degradation and forms a specialized replication vacuole. Moreover, L. pneumophila produces OMVs under various growth conditions. To understand the role of OMVs in the infection process of human macrophages, we set up a protocol to purify bacterial membrane vesicles from liquid culture. The method is based on differential ultracentrifugation. The enriched OMVs were subsequently analyzed with regard to their protein and lipopolysaccharide (LPS) amount and were then used for the treatment of a human monocytic cell line or murine bone marrow-derived macrophages. The pro-inflammatory responses of those cells were analyzed by enzyme-linked immunosorbent assay. Furthermore, alterations in a subsequent infection were analyzed. To this end, the bacterial replication of L. pneumophila in macrophages was studied by colony-forming unit assays. 
Here, we describe a detailed protocol for the purification of L. pneumophila OMVs from liquid culture by ultracentrifugation and for the downstream analysis of their pro-inflammatory potential on macrophages.

The experimental setup to prepare L. pneumophila OMVs and to analyze their influence on the pro-inflammatory response of macrophages following infection is depicted in Figure 1. The pro-inflammatory potential of the prepared OMVs can be analyzed on PMA-differentiated THP-1 cells, which is shown in Figure 2. THP-1 cells respond with a time- and dose-dependent increase of IL-8 and IL-6 secretion. Additionally, the influence of different TLRs on L. pneumophila OMV recognition can be analyzed by using mBMDM from different genetic backgrounds, as presented by the CXCL1 ELISA in Figure 3. mBMDM from WT mice secreted CXCL1 after OMV stimulation, while mBMDM TLR2/4-/- secreted significantly less. To study the impact of L. pneumophila OMVs on bacterial replication in THP-1 macrophages, cells were pre-incubated with OMVs and then additionally infected with L. pneumophila (Figure 4 A). The pre-stimulation of THP-1-derived macrophages first reduces the bacterial replication after 24 h of infection, but it leads to a doubling in CFU count at the later time point (48 h p.i.). The impact of Toll-like receptor (TLR) signaling on OMV recognition following the infection of the macrophages can be assessed by mBMDM, as presented in Figure 4 B. Bacterial replication increases by tenfold in mBMDM from WT animals after OMV pre-incubation, while TLR2-/- and TRIF/MyD88-/-cells do not show this increase in L. pneumophila replication.
<img alt="Figure 1" src="/files/ftp_upload/55146/55146fig1.jpg" /> 
Figure 1: Experimental procedure. (A) L. pneumophila Corby WT from 10-cm BCYE agar plates are used to inoculate a small liquid culture (10 mL), which is transferred into 90 mL of fresh YEB medium after 6 h. A small volume is also plated on a blood agar plate to check for purity. Bacteria are incubated at 37 &#176;C until the early stationary phase (OD600 = 3.0-3.5). (B) The liquid culture is centrifuged and sterile-filtered to remove the bacteria. The Legionella-free supernatant is then ultracentrifuged to obtain an OMV pellet, which is resuspended in PBS and ultracentrifuged again. The isolated vesicles are resuspended, checked for purity, and quantified for the protein amount. The scale bar represents 2.5 cm. (C) Human or murine macrophages are stimulated with the quantified OMVs. The cell culture supernatant can be used for ELISA, or macrophages can be infected with L. pneumophila to determine bacterial replication by CFU assay on 10-cm BCYE agar plates. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55146/55146fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55146/55146fig2.jpg" /> 
Figure 2: Pro-inflammatory activation of THP-1 cells by L. pneumophila OMVs. (A) Here, the monocytic THP-1 cell line is used as a model for alveolar macrophages. PMA-differentiated THP-1 cells were treated with increasing doses of L. pneumophila OMVs (0.01-25 &#181;g/mL) for 24 and 48 h, respectively. The cell-free supernatant was used for IL-8 ELISA. The mean values of three independent experiments &#177; SEM are shown. THP-1 cells responded to as little as 0.01 &#181;g/mL L. pneumophila OMVs with significant IL-8 secretion, which was time- and dose-dependent. (B) L. pneumophila OMVs (0.1-10 &#181;g/mL) were used to stimulate PMA-differentiated THP-1 cells. The supernatant was collected after 24 and 48 h of incubation, and the released IL-6 was measured in the supernatant via ELISA. The mean values of three independent experiments &#177; SEM are shown. THP-1 cells secreted significant amounts of IL-6, even with the lowest dose of OMVs (0.1 &#181;g/mL). The secretion of IL-6 increased with increasing OMV doses and with prolonged incubation times. Statistics: Mann-Whitney test; *p &#60; 0.05 and **p &#60; 0.01 compared to the corresponding 0 &#181;g/mL OMV. Reprinted with permission from Reference 20. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55146/55146fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55146/55146fig3.jpg" /> 
Figure 3: The pro-inflammatory activation of macrophages depends on TLR2/4. mBMDM from WT and TLR2/4-/- mice were incubated with L. pneumophila OMVs (0.1 or 1 &#181;g/mL). CXCL1 secretion was analyzed by ELISA after 24 and 48 h, respectively. The mean values &#177; SEM of three independent experiments are shown. mBMDM from WT mice responded with a dose-dependent CXCL1 secretion after L. pneumophila OMV incubation. TLR2/4-/- mBMDM secreted significantly less CXCL1 compared to WT mBMDM, and this secretion did not increase dose-dependently. Statistics: Mann-Whitney test; *p &#60; 0.05 compared to the corresponding 0 &#181;g/mL OMV; #p &#60; 0.05 compared to an equally treated WT sample. Reprinted with permission from Reference 20. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55146/55146fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/55146/55146fig4.jpg" /> 
Figure 4: L. pneumophila OMV pre-incubation increases bacterial replication in macrophages. (A) Differentiated THP-1 cells were pre-incubated with OMVs (0.1, 1, or 10 &#181;g/mL) or LPS/IFN-&#947; (200 ng/mL each) or were left untreated for control. After pre-incubation (20 h), THP-1 cells were infected with L. pneumophila Corby WT (MOI 0.5) for 2, 24, and 48 h, respectively. THP-1 cells were lysed by the addition of saponin, and the bacteria were plated on BCYE agar plates. CFUs were calculated relative to 0 &#181;g/mL OMV after every time point. The bars represent the mean values &#177; SEM of three independent experiments, each performed in technical duplicates. There were no differences in bacterial uptake (2 h post-infection (p.i.)) in comparison to not pre-treated cells. Alterations in bacterial replication were determined after 24 and 48 h, respectively. LPS/IFN-&#947; pre-treated THP-1 cells showed a reduction in bacterial load 24 h p.i. This was also observed dose-dependently for L. pneumophila OMV pre-treated cells. At the later time point (48 h p.i.), OMV pre-treated THP-1 cells showed a doubling in L. pneumophila replication, whereas LPS/IFN-&#947; pre-treated macrophages showed a further reduction of bacterial load. Statistics: Mann-Whitney test; *p &#60; 0.05 and **p &#60; 0.01 compared to the corresponding 0 &#181;g/mL OMV. (B) mBMDM from mice with different genetic backgrounds (WT, TLR2-/-, and TRIF/MyD88-/-) were pre-incubated with 0.1 &#181;g/mL L. pneumophila OMVs for 20 h and were then infected with a flagellin-deficient mutant of L. pneumophila Corby (MOI 0.5) for 48 h. mBMDM were lysed by the addition of saponin, and the Legionella were plated on BCYE agar plates. The CFU were calculated relative to 0 &#181;g/mL OMV, indicated by the solid line. The bars represent the mean values &#177;&#177; SEM of three independent experiments, each performed in duplicates. mBMDM from WT mice showed an increase in L. pneumophila replication after OMV pre-treatment. TLR2-/- macrophages showed significantly reduced Legionella replication, which was comparable to TRIF/MyD88-/- mBMDM. Statistics: Mann-Whitney test; p &#60; 0.05 compared to the WT sample. Reprinted with permission from Reference 20. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55146/55146fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages at JoVE.com

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Here, we describe the purification of Legionella pneumophila (L. pneumophila) outer membrane vesicles (OMVs) from liquid cultures. These purified vesicles are then used for the treatment of macrophages to analyze their pro-inflammatory potential.

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or ...

legionella-pneumophila-outer-membrane-vesicles-isolation-analysis

Nanyang Technological University

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JoVE Journal

Immunology and Infection

11.1K Views.  Philipps-University Marburg. Here, we describe the purification of Legionella pneumophila (L. pneumophila) outer membrane vesicles (OMVs) from liquid cultures. These purified vesicles are then used for the treatment of macrophages to analyze their pro-inflammatory potential.

Video: Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and replicates within alveolar macrophages. Its virulence depends on the Dot/Icm type IV secretion system (T4SS), which is essential to establish a replication permissive vacuole known as the Legionella containing vacuole (LCV). L. pneumophila infection can be modeled in mice however most mouse strains are not permissive, leading to the search for novel infection models. We have recently shown that the larvae of the wax moth Galleria mellonella are suitable for investigation of L. pneumophila infection. G. mellonella is increasingly used as an infection model for human pathogens and a good correlation exists between virulence of several bacterial species in the insect and in mammalian models. A key component of the larvae&#39;s immune defenses are hemocytes, professional phagocytes, which take up and destroy invaders. L. pneumophila is able to infect, form a LCV and replicate within these cells. Here we demonstrate protocols for analyzing L. pneumophila virulence in the G. mellonella model, including how to grow infectious L. pneumophila, pretreat the larvae with inhibitors, infect the larvae and how to extract infected cells for quantification and immunofluorescence microscopy. We also describe how to quantify bacterial replication and fitness in competition assays. These approaches allow for the rapid screening of mutants to determine factors important in L. pneumophila virulence, describing a new tool to aid our understanding of this complex pathogen.

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

The larva of the wax moth Galleria mellonella was recently established as an in vivo model to study Legionella pneumophila infection. Here, we demonstrate fundamental techniques to characterize the pathogenesis of Legionella in the larvae, including inoculation, measurement of bacterial virulence and replication as well as extraction and analysis of infected hemocytes.

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and ...

Isolation of Human Monocytes by Double Gradient Centrifugation and Their Differentiation to Macrophages in Teflon-coated Cell Culture Bags

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to understand the underlying mechanisms of these diseases. We therefore present a straightforward protocol for the isolation of human monocytes from buffy coats, followed by a differentiation procedure which results in high macrophage yields. The technique relies mostly on commonly available lab equipment and thus provides a cost and time effective way to obtain large quantities of human macrophages. Briefly, buffy coats from healthy blood donors are subjected to a double density gradient centrifugation to harvest monocytes from the peripheral blood. These monocytes are then cultured in fluorinated ethylene propylene (FEP) Teflon-coated cell culture bags in the presence of macrophage colony-stimulating factor (M-CSF). The differentiated macrophages can be easily harvested and used for subsequent studies and functional assays. Important methods for quality control and validation of the isolation and differentiation steps will be highlighted within the protocol. In summary, the protocol described here enables scientists to routinely and reproducibly isolate human macrophages without the need for cost intensive tools. Furthermore, disease models can be studied in a syngeneic human system circumventing the use of murine macrophages.

We present a simple and efficient protocol for the generation of human macrophages. Buffy coats are processed by double density gradient centrifugation and isolated monocytes are then differentiated to macrophages in Teflon-coated cell culture bags. This maximizes macrophage yields and facilitates cell harvesting for subsequent experiments.

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to ...

Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. Macrophages express surface Toll-like receptors (TLRs), which recognize molecular patterns conserved through evolution in a wide range of microorganisms. TLRs play a central role in macrophage activation which is usually associated with gene expression alteration. Macrophages are critical in many diseases and have emerged as attractive targets for therapy. In the following protocol, we describe a procedure to isolate murine peritoneal macrophages using Brewer&#8217;s thioglycollate medium. The latter will boost monocyte migration into the peritoneum, accordingly this will raise macrophage yield by 10-fold. Several studies have been carried out using bone marrow, spleen or peritoneal derived macrophages. However, peritoneal macrophages were shown to be more mature upon isolation and are more stable in their functionality and phenotype. Thus, macrophages isolated from murine peritoneal cavity present an important cell population that can serve in different immunological and metabolic studies. Once isolated, macrophages were stimulated with different TLR ligands and consequently&#160;gene expression was evaluated.

We describe here a simple protocol to isolate murine peritoneal macrophages. This procedure is followed by RNA extraction to carry out gene expression analysis upon Toll-like receptors stimulation.

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. ...

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 of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

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 ...

Quantification of Monocyte Chemotactic Activity In Vivo and Characterization of Blood Monocyte Derived Macrophages

Tissue homeostasis and repair are critically dependent on the recruitment of monocyte-derived macrophages. Both under- and over-recruitment of monocyte-derived macrophages can impair wound healing. We showed that high fat and high sugar diets promote monocyte priming and dysfunction, converting healthy blood monocytes into a hyper chemotactic phenotype poised to differentiate into macrophages with dysregulated activation profiles and impaired phenotypic plasticity. The over-recruitment of monocyte-derived macrophages and recruitment of macrophages with dysregulated activation profiles is believed to be a major contributor to the development of chronic inflammatory diseases associated with metabolic disorders, including atherosclerosis and obesity. The goal of this protocol is to quantify the chemotactic activity of blood monocytes as a biomarker for monocyte priming and dysfunction and to characterize the macrophage phenotype blood monocytes are poised to differentiate into in these mouse models. Using single cell Western blot analysis, we show that after 24 h 33%of cells recruited into MCP-1-loaded basement membrane-derived gel plugs injected into mice are monocytes and macrophages; 58% after day 3. However, on day 5, monocyte and macrophage numbers were significantly decreased. Finally, we show that this assays also allows for the isolation of live macrophages from the surgically retrieved basement membrane-derived gel plugs, which can then be subjected to subsequent characterization by single cell Western blot analysis.

Here we present a protocol to quantify the chemotactic activity of blood monocytes in mouse models, to assess the effects of nutritional, pharmacological and genetic interventions on blood monocyte and to characterize the blood monocytes derived macrophages in mouse models using monocyte-chemoattractant protein-1 (MCP-1)-loaded basement membrane-derived gel plugs.

Tissue homeostasis and repair are critically dependent on the recruitment of monocyte-derived macrophages. Both under- and over-recruitment of ...

Experimental Analysis of Apoptotic Thymocyte Engulfment by Macrophages

Cell apoptosis is a natural process and plays a critical role in embryonic development, homeostatic regulation, immune tolerance induction, and resolution of inflammation. Accumulation of apoptotic debris in the body may trigger chronic inflammatory responses that lead to systemic autoimmune diseases over time. Impaired apoptotic cell clearance has been implicated in a variety of autoimmune diseases. Apoptotic clearance is a complex process rarely detected under physiological conditions. It involves abundant surface receptors and signaling molecules. Studying the process of apoptotic cell clearance provides insightful molecular mechanisms and subsequent biological responses, which may lead to the development of new therapeutics. Here, we describe protocols for the induction of apoptotic thymocytes, the preparation of peritoneal macrophages, and the analysis of apoptotic cell clearance by flow cytometry and microscopy. All cells will undergo apoptosis at a certain stage, and many residential and circulating cells can uptake apoptotic debris. Therefore, the protocol described here can be used in many applications to characterize apoptotic cell binding and ingestion by many other cell types.

Here, we present a protocol to prepare apoptotic thymocytes and peritoneal macrophages and analyze the efficiency of efferocytosis and the specific inhibitor-mediated blocking of apoptotic thymocytes engulfment. This protocol has a broad application in cell-mediated clearance of other particles including artificial beads and bacteria.

Cell apoptosis is a natural process and plays a critical role in embryonic development, homeostatic regulation, immune tolerance induction, and resolution ...