Research in the Robinson&#39;s lab is supported by funding from Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases, University of Cologne, Germany (CECAD; funded by the DFG within the Excellence Initiative of the German federal and state governments) and grants from Deutsche Forschungsgemeinschaft (SFB 670), K&#246;ln Fortune, and Maria-Pesch Foundation of the University of Cologne, Germany.

All the steps involving the use of pathogenic S. typhimurium must be carried out in a BSL-2 or higher biological security level facility. The culture and coating of S. Typhimurium, as well as the infection of bone marrow-derived macrophages (BMDMs) must be performed under a laminar flow hood to prevent contamination. The isolation of S. typhimurium-containing phagosomes can be performed on any BSL-2 laboratory bench.
The extraction of bone marrow from mice for its differentiation into macrophages was performed in accordance with institutional guidelines on animal welfare and approved by the North Rhine-Westphalian State Agency for Nature, Environment, and Consumer Protection [Landesamt f&#252;r Natur, Umwelt and Verbraucherschutz (LANUV) Nordrhein-Westfalen; File no: 84-02.05.40.14.082 and 84-02.04.2015.A443] and the University of Cologne.
1. Culturing S. typhimurium
<ol>
	<li>Inoculate S. typhimurium (SL1344 strain) from a single bacterial colony into 5 mL of brain heart infusion (BHI) broth using a bacterial loop.</li>
	<li>Incubate the bacterial suspension in an incubator at 37 &#176;C overnight with shaking.</li>
	<li>On the following day, transfer 1 mL of the bacterial suspension into 19 mL of BHI broth in a conical flask and incubate at 37 &#176;C in an incubator with shaking.</li>
	<li>Monitor the S. typhimurium growth by measuring the optical density (OD) at 600 nm (OD600) with a spectrophotometer. Measurements should be taken approximately every 30 min.</li>
	<li>When OD600 reaches 1.0, remove bacterial suspension from the incubator and transfer into 50 mL tube. Centrifuge bacteria at 5,400 x g for 15 min at 4 &#176;C.</li>
	<li>Remove supernatant and resuspend in 10 mL of sterile phosphate-buffered saline (PBS).</li>
	<li>Centrifuge at 5,400 x g for 15 min at 4 &#176;C.</li>
	<li>Remove the supernatant and resuspend the bacteria in 4.9 mL of sterile PBS.</li>
</ol>
2. Coating of S. typhimurium with NHS-Biotin/Streptavidin-conjugated Magnetic Beads
<ol>
	<li>Add 100 &#181;L of 10 mg/mL biotin linking solution (NHS-Biotin dissolved in dimethyl sulfoxide, DMSO) freshly prepared, to the bacterial suspension prepared in the previous step. For example, dilute 5 mg of NHS-Biotin into 0.5 mL of sterile DMSO. Mix properly by pipetting up and down several times.</li>
	<li>Split the mix into five 1.5 mL tubes.</li>
	<li>Incubate for 2 h at room temperature (RT) on a thermoblock with constant shaking at 350 rpm.</li>
	<li>Centrifuge tubes at 15,000 x g for 10 min at RT and discard the supernatant.</li>
	<li>Resuspend in 1 mL of sterile PBS, centrifuge at 15,000 x g for 10 min at RT and discard the supernatant.</li>
	<li>Repeat step 2.5 two more times to completely remove the excess biotin linking solution.</li>
	<li>After the last wash, resuspend the pellet in 1 mL of sterile PBS.</li>
	<li>Transfer 100 &#181;L of 10 mg/mL streptavidin-conjugated magnetic beads solution into a 1.5 mL tube and leave it on the magnetic rack for 5 min.</li>
	<li>Remove the solvent with a pipette, take the streptavidin- conjugated magnetic beads solution from the magnetic rack, and resuspend it with 1 mL of biotin coated-bacterial suspension prepared in step 2.7.</li>
	<li>Incubate for 1 h at RT on a thermoblock with constant shaking at 350 rpm.</li>
	<li>Place the solution on the magnetic rack; wait for 5 min and then remove with a pipette the bacteria that did not adhere onto the wall (keep it in a tube labeled as &#34;non-coated bacteria&#34;). The fraction adhering to the wall of the tube in contact with the magnet is the biotin/streptavidin-coated bacteria.</li>
	<li>Remove the tube containing the labeled bacteria from the magnetic rack and resuspend in 1 mL of sterile PBS.</li>
	<li>Place it again on the magnetic rack, wait for 5 min, and use a pipette to remove the PBS.</li>
	<li>Repeat steps 2.13 and 2.14 two more times to remove all the bacteria that are not coated with the streptavidin-conjugated magnetic beads.</li>
	<li>After the last wash, resuspend the coated-bacteria in 500 &#181;L of sterile PBS and label the tube as &#34;coated bacteria&#34;.</li>
	<li>Count the colony forming units (cfu) of both &#34;non-coated bacteria&#34; and &#34;coated bacteria&#34; solutions by plating serial dilutions on BHI agar plates. Approximately 2 x 108 cfu/mL of coated-bacteria are obtained from 20 mL of initial bacterial suspension with OD600 of 1.0. Keep the coated-bacterial suspension at 4 &#176;C to be used on the following day to infect macrophages.</li>
</ol>
3. Infection of BDMDs
<ol>
	<li>On the same day when the bacteria are coated with biotin and streptavidin-conjugated magnetic beads, plate fully the differentiated BMDMs22 in 6 cm dishes at a density of 5 x 106 cells per dish.</li>
	<li>On the next day, centrifuge the coated-bacterial suspension at 15,000 x g for 5 min at 4 &#176;C.</li>
	<li>Remove the supernatant and resuspend in Roswell Park Memorial Institute (RPMI) medium supplemented with 10% fetal bovine serum (FBS) at a concentration of 10 x 106 cfu/mL.</li>
	<li>To infect macrophages at a multiplicity of infection (MOI) of 10, remove the medium from the BMDMs and add 5 mL of coated-bacterial suspension prepared. Optimal MOI may be assessed for every cell type or experimental purpose of the isolated phagosomes.</li>
	<li>Incubate at RT for 10 min to synchronize phagocytosis.</li>
	<li>Incubate at 37 &#176;C in a 5% CO2 incubator for 30 min to initiate phagocytosis. Other cell types may require different incubation times to ensure internalization of the bacteria.</li>
	<li>Remove the bacteria-containing medium from the dishes and wash the cells with RPMI three times to remove non-internalized bacteria.</li>
	<li>Finally add 10 mL of RPMI containing 10% FBS and 50 &#181;g/mL gentamycin to kill any non-phagocytized bacteria.</li>
	<li>Incubate the infected BMDMs at 37 &#176;C with 5% CO2 until the desired time point. The desired time point must be experimentally determined according to the bacteria and cell type used and the purpose of analysis. For the study of early phagosome-endosome fusion events in S. Typhimurium-infected BMDMs, a 30 min incubation is recommended. For the analysis of later events, phagosomes can be extracted after 2 h or 4 h of incubation. Prolonged incubation of macrophages with S. Typhimurium beyond 24 h results in death of the macrophages. Extended intracellular infection before the phagosome extraction could probably lead to degradation of biotin molecules. However, we did not observe loss of biotin on coated-bacteria until 24 h.</li>
</ol>
4. Isolation of S. typhimurium-containing Phagosomes
<ol>
	<li>Prepare the volume of required phagosome isolation buffer A (50 mM PIPES, pH.7, 50 mM MgCl2, 5 mM EGTA) by adding dithiothreitol (DTT) to a final concentration of 1 mM, Cytochalasin B to a final concentration of 10 &#181;M, and protease and phosphatase inhibitors as recommended by the manufacturer. 
	NOTE: DTT and Cytochalasin B must be added to the phagosome isolation buffer A always immediately before use. Cytochalasin B disrupts the cytoskeleton, facilitating the rupture of the cell membrane.</li>
	<li>At the desired time point, remove the medium from the infected cells and wash with sterile PBS warmed to RT.</li>
	<li>Add 750 &#181;L of phagosome isolation buffer A per dish and incubate 20 min on ice. When using different cell numbers, the volume of isolation buffer A should be adjusted proportionally.</li>
	<li>Add 250 &#181;L of phagosome isolation buffer B (50 mM PIPES, pH.7, 50 mM MgCl2, 5 mM EGTA, 220 mM Mannitol, and 68 mM sucrose).Rock the plate to ensure that the buffer reaches the complete surface of the dish. When using different cell numbers, the volume of isolation buffer B should be adjusted proportionally.</li>
	<li>Remove the cells from the dish by gently scraping using a rubber policeman and transfer them to a pre-chilled 1.5 mL tube.</li>
	<li>Pass the cell suspension through a 26G needle using a 1 mL syringe at least 15 times (one aspiration plus one ejection of the cell suspension counts as one time). This is enough to release the cytosolic content of BMDMs. The number of passes through the needle must be optimized for every cell type.</li>
	<li>Place the cell suspension on the magnetic rack and wait 5 min. The particles attached to the magnet are the phagosomes containing coated-S. Typhimurium. The suspension contains the rest of the cellular components.</li>
	<li>Transfer the suspension into a 1.5 mL tube labeled as &#34;cytosol&#34;.</li>
	<li>Remove the tube with the isolated phagosomes from the magnetic rack and resuspend them in 1 mL of sterile PBS.</li>
	<li>Place the phagosome suspension on the magnetic rack, wait for 5 min, and remove the PBS.</li>
	<li>Repeat steps 4.9 and 4.10 to wash the isolated S. typhimurium-containing phagosomes.</li>
	<li>Finally remove the PBS and resuspend the phagosomes in the required buffer; for example, in a radioimmunoprecipitation assay (RIPA) buffer for protein analysis. Approximately 50-200 &#181;g of protein is obtained per sample following this protocol, depending on the initial amount of cells and the MOI of infection.</li>
</ol>

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The authors have nothing to disclose.

A new method for the isolation of S. typhimurium-containing phagosomes by coating the bacteria with biotin and streptavidin-conjugated magnetic beads is described here. After gentle disruption of the cell membrane, bacteria-containing phagosomes can be easily extracted using a magnetic rack. We show that labeling of the bacteria preserves the capacity of the pathogen to induce inflammation and does not alter the phagocytic property of the host cell. Importantly, phagosomes obtained by this method are en...

Macrophages are circulating specialized phagocytic cells that detect, engulf, and degrade any foreign particle present in peripheral tissues, ranging from apoptotic cells to invading microorganisms such as bacteria. Upon surface receptor-mediated recognition of pathogen specific markers commonly present on the surface of microorganisms (known as pathogen-associated molecular patterns or PAMPs), macrophages initiate a complex reorganization of cellular membrane in order to surround and phagocytize the pathogen1.
The engulfed pathogen is then contained by the macrophage in an intracellular vesicle known as phagosome. Through a series of fusion and fission events with other vesicles such as endosomes and lysosomes, the pathogen-containing phagosome acquires a set of proteins required for the elimination of the phagosomal content. Therefore, the enzymatic composition of the phagosome is highly variable during the course of this process, known as phagosome maturation2.
Shortly after phagocytosis, the multimeric complex vacuolar ATPase (v-ATPase) is incorporated into the phagosome membrane by fusion with endosomes3. This complex utilizes ATP to pump protons from the cytosol to the lumen of the phagosome4. Acidification of the phagosome is essential for the fusion events with other vesicles5 and for the activation of a great number of pH-dependent degradative enzymes6. Another multimeric enzymatic complex that is quickly assembled on the phagosome membrane is the NADPH-oxidase (NOX) complex. NOX complex oxidizes NADPH in order to produce reactive oxygen species (ROS) that are secreted into the phagosome lumen and that significantly contribute to the killing of the engulfed microorganisms7.
During the initial steps of maturation, phagosomes present markers typically such as Rab5 and Rab7 of early and late endosomes respectively along with the V0 subunit of the v-ATPase8. Fusion of phagosomes with lysosomes and late endosomes results in the exposure of the phagocytized pathogen to a wide variety of hydrolytic enzymes such as cathepsin proteases, lipases, and &#946;-galactosidase9. Acidification of the lumen is also required for the activation of these enzymes. For example, the cleavage of cathepsin D to produce the active short form is pH-dependent10. These enzymes degrade the pathogen and mediate the production of pathogen-derived short peptides that are presented by the macrophage major histocompatibility complex (MHC) class II molecules to T cells to trigger an adaptive immune response11.
Hence, phagosome maturation is crucial for the innate immune response and links the innate and adaptive arms of the immune system. It is no surprise that the pathogens have evolved strategies to overcome elimination by macrophages through the above-described process of phagosome maturation. For example, the intracellular bacteria Mycobacterium tuberculosis and Legionella pneumophila prevent phagosome maturation by inhibiting v-ATPase assembly and consequent lumen acidification12,13. Other bacteria, such as Listeria monocytogenes or Shigella flexneri induce pore formation in the phagosome membrane to escape into the cytosol14,15. On the other hand, Salmonella enterica serovar typhimurium (S. typhimurium) is able to modify the properties of the phagosome within the vacuole to transform it into a suitable location for its replication16. This ability makes S. typhimurium a very interesting model to study pathogen-mediated interference of phagosome maturation.
S. 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 within phagosomes17. Some reports have previously described that S. typhimurium-containing phagosomes present makers for both endosomes and lysosomes18, and other studies have found phagosome-lysosome fusion prevented upon S. Typhimurium infection19.
Initially, phagosome maturation upon S. typhimurium infection has been investigated by immunofluorescence microscopy. The development of techniques for the isolation of bacteria-containing phagosomes enabled a more accurate study of the phagosome content in terms of endosome and lysosome markers. To date, the main method used for the isolation of bacteria-containing phagosomes is the subcellular fractionation on sucrose step gradients18,20. However, this method requires multiple centrifugation steps that can cause mechanical damage to phagosomes, can affect the stability of phagosomal components (proteins and lipids), and is time consuming. Moreover, it requires the use of an ultracentrifuge: a piece of specialized equipment that is not accessible for every laboratory.
Recently, a new approach has been applied to the isolation of bacterial-containing phagosomes, in which bacterial pathogens are labeled with biotinylated lipopetide (Lipobiotin) and later extracted using streptavidin-conjugated magnetic beads21. We propose an alternative complementary method by labeling bacterial surface amine-containing macromolecules with NHS-Biotin followed by streptavidin-conjugated magnetic beads. Phagosomes obtained by this method are highly enriched in endosome and lysosome markers and can be used for a broad range of assays, from protein analysis to omics analysis. Additionally, it does not require specialized equipment such as ultracentrifuges. Moreover, by eliminating the centrifugation steps, both the mechanical damage to phagosomes and the amount of time employed are considerably reduced. This method can be easily adapted for the isolation of phagosomes containing other bacteria, such as the Gram-positive Staphylococcus aureus, also included in this manuscript. In summary, this method for the isolation of S. typhimurium-containing phagosomes is a simple, cost-effective, and less time consuming than the classical isolation by sucrose gradient-ultracentrifugation, rendering highly enriched bacteria-containing phagosomes.

<table><tbody><tr><td>EZ-Link NHS Biotin</td><td>Thermo Fisher Scientific</td><td>20217</td><td></td></tr><tr><td>FluidMag Streptavidin</td><td>Chemicell</td><td>4205</td><td></td></tr><tr><td>PIPES</td><td>Carl Roth</td><td>9156.2</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Carl Roth</td><td>A537.4</td><td></td></tr><tr><td>EGTA</td><td>Carl Roth</td><td>3054.3</td><td></td></tr><tr><td>Sucrose</td><td>Carl Roth</td><td>4621.1</td><td></td></tr><tr><td>Mannitol</td><td>Carl Roth</td><td>4175.1</td><td></td></tr><tr><td>DTT</td><td>Sigma</td><td>43816</td><td></td></tr><tr><td>Halt Protease and Phosphatase inhibitor cocktail</td><td>Thermo Fisher Scientific</td><td>1861280</td><td></td></tr><tr><td>Cytochalasin B</td><td>Sigma</td><td>C6762</td><td></td></tr><tr><td>DYNAL or DynaMag Magnet</td><td>Thermo Fisher Scientific</td><td>12321D</td><td></td></tr><tr><td>SmartSpec 3000 Spectrophotometer</td><td>Bio-Rad</td><td>170-2501</td><td></td></tr><tr><td>Bacterial loop (10&amp;micro;l)</td><td>Sarstedt</td><td>86.1562.010</td><td></td></tr><tr><td>Salmonella enterica serovar Typhimurium SL1344</td><td>Leibniz Institute DSMZ-German collection of Microorganisms and Cell Cultures</td><td></td><td></td></tr><tr><td>RPMI</td><td>Biochrom</td><td>FG1415</td><td></td></tr><tr><td>PBS</td><td>Biochrom</td><td>L1825</td><td></td></tr><tr><td>Cy5-streptavidin</td><td>Invitrogen</td><td>SA1011</td><td></td></tr><tr><td>anti-beta-actin antibody</td><td>Santa Cruz Biotechnology</td><td>sc-47778</td><td></td></tr><tr><td>anti-mCherry antibody</td><td>Thermo Fisher Scientific</td><td>PA5-34974</td><td></td></tr><tr><td>anti-Rab5 antibody</td><td>Santa Cruz Biotechnology</td><td>sc-46692</td><td></td></tr><tr><td>anti-Rab7 antibody</td><td>Santa Cruz Biotechnology</td><td>sc-10764</td><td></td></tr><tr><td>anti-v-ATPase (V0) antibody</td><td>Santa Cruz Biotechnology</td><td>sc-28801</td><td></td></tr><tr><td>anti-v-ATPase (V1) antibody</td><td>Santa Cruz Biotechnology</td><td>sc-20943</td><td></td></tr><tr><td>anti-cathepsin D antibody</td><td>Santa Cruz Biotechnology</td><td>sc-6486</td><td></td></tr><tr><td>anti-Tomm20 antibody</td><td>Santa Cruz Biotechnology</td><td>sc-17764</td><td></td></tr><tr><td>anti-calnexin antibody</td><td>Santa Cruz Biotechnology</td><td>sc-46669</td><td></td></tr><tr><td>anti-GAPDH antibody</td><td>Santa Cruz Biotechnology</td><td>sc-20357</td><td></td></tr></tbody></table>

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 bacteria-containing phagosomes by this protocol requires the biotinylation of the bacteria as a first step. We therefore assessed the effectiveness of S. typhimurium biotinylation by confocal microscopy analysis of BMDMs infected with biotinylated mCherry-S. typhimurium labeled with Cy5-Streptavidin. Briefly, BMDMs were infected as described in this protocol with mCherry-S. typhimurium previously biotinylated but not incub...

Watch this Scientific Journal Video about Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages at JoVE.com

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.

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. ...

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JoVE Journal

Immunology and Infection

10.6K Views.  University of Cologne. 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.

Video: Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although the isolation methods of liver macrophages have been well-described4-6, most of these methods require sophisticated equipment, such as a centrifugal elutriator and technical skills. Here, we provide a novel method to obtain liver macrophages in sufficient number and purity from mixed primary cultures of adult rat liver cells, as schematically illustrated in Figure 1.
After dissociation of the liver cells by two-step perfusion method7,8,a fraction mostly composed of parenchymal hepatocytes is prepared and seeded into T75 tissue culture flasks with culture medium composed of DMEM and 10% FCS.Parenchymal hepatocytes lose the epithelial cell morphology within a few days in culture, degenerate or transform into fibroblast-like cells (Figure 2). As the culture proceeds, around day 6, phase contrast-bright, round macrophage-like cells start to proliferate on the fibroblastic cell sheet (Figure 2). The growth of the macrophage-like cells continue and reach to maximum levels around day 12, covering the cell sheet on the flask surface. By shaking of the culture flasks, macrophages are readily suspended into the culture medium. Subsequent transfer and short incubation in plastic dishes result in selective adhesion of macrophages(Figure 3), where as other contaminating cells remain suspended. After several rinses with PBS, attached macrophages are harvested. More than 106 cells can be harvested repeatedly from the same T75 tissue culture flask at two to three day intervals for more than two weeks(Figure 3).The purities of the isolated macrophages were 95 to 99%, as evaluated by flow cytometry or immunocytochemistry with rat macrophage-specific antibodies (Figure 4).The isolated cells show active phagocytosis of polystylene beads (Figure 5), proliferative response to recombinant GM-CSF, secretion of inflammatory/anti-inflammatory cytokines upon stimulation with LPS, and formation of multinucleated giant cells9.
In conclusion, we provide a simple and efficient method to obtain liver macrophages in sufficient number and purity without complex equipment and skills.This method might be applicable to other mammalian species.

A novel method to obtain macrophages from primary culture of rat liver cells is described. This method utilizes the proliferation of macrophages in the culture, followed by shaking of culture flasks and purification by selective attachment to plastic dishes. This technique efficiently provides liver macrophages without complex equipment and skills.

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although ...

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

The opportunistic pathogen Legionella pneumophila is an amoeba-resistant bacterium, which also replicates in alveolar macrophages thus causing the severe pneumonia "Legionnaires' disease"1. In protozoan and mammalian phagocytes, L. pneumophila employs a conserved mechanism to form a specific, replication-permissive compartment, the "Legionella-containing vacuole" (LCV). LCV formation requires the bacterial Icm/Dot type IV secretion system (T4SS), which translocates as many as 275 "effector" proteins into host cells. The effectors manipulate host proteins as well as lipids and communicate with secretory, endosomal and mitochondrial organelles2-4.
The formation of LCVs represents a complex, robust and redundant process, which is difficult to grasp in a reductionist manner. An integrative approach is required to comprehensively understand LCV formation, including a global analysis of pathogen-host factor interactions and their temporal and spatial dynamics. As a first step towards this goal, intact LCVs are purified and analyzed by proteomics and lipidomics.
The composition and formation of pathogen-containing vacuoles has been investigated by proteomic analysis using liquid chromatography or 2-D gel electrophoresis coupled to mass-spectrometry. Vacuoles isolated from either the social soil amoeba Dictyostelium discoideum or mammalian phagocytes harboured Leishmania5, Listeria6, Mycobacterium7, Rhodococcus8, Salmonella9 or Legionella spp.10. However, the purification protocols employed in these studies are time-consuming and tedious, as they require e.g. electron microscopy to analyse LCV morphology, integrity and purity. Additionally, these protocols do not exploit specific features of the pathogen vacuole for enrichment.
The method presented here overcomes these limitations by employing D. discoideum producing a fluorescent LCV marker and by targeting the bacterial effector protein SidC, which selectively anchors to the LCV membrane by binding to phosphatidylinositol 4-phosphate (PtdIns(4)P)3,11 . LCVs are enriched in a first step by immuno-magnetic separation using an affinity-purified primary antibody against SidC and a secondary antibody coupled to magnetic beads, followed in a second step by a classical Histodenz density gradient centrifugation12,13 (Fig. 1).
A proteome study of isolated LCVs from D. discoideum revealed more than 560 host cell proteins, including proteins associated with phagocytic vesicles, mitochondria, ER and Golgi, as well as several GTPases, which have not been implicated in LCV formation before13. LCVs enriched and purified with the protocol outlined here can be further analyzed by microscopy (immunofluorescence, electron microscopy), biochemical methods (Western blot) and proteomic or lipidomic approaches.

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

This article describes a method for the isolation and purification of intact Legionella-containing vacuoles (LCVs) from amoeba and macrophages. The two-step protocol comprises LCV enrichment by immuno-magnetic separation using an antibody against a bacterial LCV marker and further purification by density gradient centrifugation.

The opportunistic pathogen Legionella pneumophila is an amoeba-resistant bacterium, which also replicates in alveolar macrophages thus causing the severe ...

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying Salmonella-host interactions are&#160;important to elucidate the molecular pathogenesis of Salmonella infection. The Gentamicin protection assay to phenotype Salmonella association, invasion and replication in phagocytic cells was adapted to allow high-throughput screening to define the roles of deletion mutants of Salmonella enterica serotype Typhimurium in host interactions using RAW 264.7 murine macrophages. Under this protocol, the variance in measurements is significantly reduced compared to the standard protocol, because wild-type and multiple mutant strains can be tested in the same culture dish and at the same time. The use of multichannel pipettes increases the throughput and enhances precision. Furthermore, concerns related to using less host cells per well in 96-well culture dish were addressed. Here, the protocol of the modified in vitro Salmonella invasion assay using phagocytic cells was successfully employed to phenotype 38 individual Salmonella deletion mutants for association, invasion and intracellular replication. The in vitro phenotypes are presented, some of which were subsequently confirmed to have in vivo phenotypes in an animal model. Thus, the modified, standardized assay to phenotype Salmonella association, invasion and replication in macrophages with high-throughput capacity could be utilized more broadly to study bacterial-host interactions.

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

A high-throughput assay to in vitro phenotype Salmonella or other bacterial association, invasion, and replication in phagocytic cells with high-throughput capacity was developed. The method was employed to evaluate Salmonella gene knockout mutant strains for their involvements in host-pathogen interactions.

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying ...

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. ...

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

Intracellular bacterial pathogens can replicate in the cytosol or in specialized pathogen-containing vacuoles (PCVs). To reach the cytosol, bacteria like Shigella flexneri and Francisella novicida need to induce the rupture of the phagosome. In contrast, Salmonella typhimurium replicates in a vacuolar compartment, known as Salmonella-containing vacuole (SCV). However certain mutants of Salmonella fail to maintain SCV integrity and are thus released into the cytosol. The percentage of cytosolic vs. vacuolar bacteria on the level of single bacteria can be measured by differential permeabilization, also known as phagosome-protection assay. The approach makes use of the property of detergent digitonin to selectively bind cholesterol. Since the plasma membrane contains more cholesterol than other cellular membranes, digitonin can be used to selectively permeabilize the plasma membrane while leaving intracellular membranes intact. In brief, following infection with the pathogen expressing a fluorescent marker protein (e.g. mCherry among others), the plasma membrane of host cells is permeabilized with a short incubation in digitonin containing buffer. Cells are then washed and incubated with a primary antibody (coupled to a fluorophore of choice) directed against the bacterium of choice (e.g. anti-Salmonella-FITC) and washed again. If unmarked bacteria are used, an additional step can be done, in which all membranes are permeabilized and all bacteria stained with a corresponding antibody. Following the staining, the percentage of vacuolar and cytosolic bacteria can be quantified by FACS or microscopy by counting single or double-positive events. Here we provide experimental details for use of this technique with the bacterium Salmonella typhimurium. The advantage of this assay is that, in contrast to other assay, it provides a quantification on the level of single bacteria, and if analyzed by microscopy provides the exact number of cytosolic and vacuolar bacteria in a given cell.

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

We describe the quantification of cytosolic and vacuolar Salmonella typhimurium in bone-marrow derived macrophages using differential digitonin permeabilization.

Intracellular bacterial pathogens can replicate in the cytosol or in specialized pathogen-containing vacuoles (PCVs). To reach the cytosol, bacteria like ...

"Phagosome Closure Assay" to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy (TIRFM)

Phagocytosis is a mechanism used by specialized cells to internalize and eliminate microorganisms or cellular debris. It relies on profound rearrangements of the actin cytoskeleton that is the driving force for plasma membrane extension around the particle. In addition, efficient engulfment of large material relies on focal exocytosis of intracellular compartments. This process is highly dynamic and numerous molecular players have been described to have a role during phagocytic cup formation. The precise regulation in time and space of all of these molecules, however, remains elusive. In addition, the last step of phagosome closure has been very difficult to observe because inhibition by RNA interference or dominant negative mutants often results in stalled phagocytic cup formation. 
We have set up a dedicated experimental approach using total internal reflection fluorescence microscopy (TIRFM) combined with epifluorescence to monitor step by step the extension of pseudopods and their tips in a phagosome growing around a particle loosely bound to a coverslip. This method allows us to observe, with high resolution the very tips of the pseudopods and their fusion during closure of the phagosome in living cells for two different fluorescently tagged proteins at the same time.

"Phagosome Closure Assay" to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy TIRFM

We describe an experimental setup to visualize with unprecedented high resolution phagosome formation and closure in three dimensions in living macrophages, using total internal reflection fluorescence microscopy. It allows monitoring of the base of the phagocytic cup, the extending pseudopods, as well as the precise site of phagosome scission.

Phagocytosis is a mechanism used by specialized cells to internalize and eliminate microorganisms or cellular debris. It relies on profound rearrangements ...

Characterizing Salmonella Typhimurium-induced Septic Peritonitis in Mice

Sepsis is a dysregulated host immune response to microbial invasion or tissue damage, leading to organ injury at a site distant from that of the infection or damage. Currently, the widely used mice models of sepsis include lipopolysaccharide (LPS)-induced endotoxemia, cecal ligation and puncture (CLP), and monobacterial infection model systems. This protocol describes a method to study the host responses during Salmonella Typhimurium infection-induced septic peritonitis in mice. S. Typhimurium, a Gram-negative intracellular pathogen, causes typhoid-like disease in mice.
This protocol elaborates the culture preparation, induction of septic peritonitis in mice through intraperitoneal injection, and methods to study systemic host responses. Furthermore, the assessment of bacterial burden in different organs and the flow cytometric analysis of increased neutrophil numbers in the peritoneal lavage is presented. Salmonella Typhimurium-induced sepsis in mice leads to an increase in proinflammatory cytokines and rapid infiltration of neutrophils in the peritoneal cavity, leading to lower survival.
Every step in this protocol has been optimized, resulting in high reproducibility of the pathogenesis of septic peritonitis. This model is useful for studying immunological responses during bacterial sepsis, the roles of different genes in disease progression, and the effects of drugs to attenuate sepsis.

Characterizing Salmonella Typhimurium-induced Septic Peritonitis in Mice

This protocol describes the induction of Gram-negative monobacterial sepsis in a mouse model system. The model is useful in investigating the inflammatory and lethal host responses during sepsis.

Sepsis is a dysregulated host immune response to microbial invasion or tissue damage, leading to organ injury at a site distant from that of the infection ...

Using Bacterial LPS to Eliminate Bacteriophages from Salmonella Cultures

Bacteriophage Removal from Infected Salmonella Cultures

Bacteriophages, or simply phages, play a vital role in microbial environments, impacting bacterial populations and shaping their evolution and interactions. These organisms are viruses that infect and replicate within bacterial hosts. Phages are ubiquitous on Earth, highly diverse, and very abundant. While bacteriophages have valuable roles in different environments and are a key area of research in microbiology and ecology, their presence can be undesirable in certain industrial processes or products. Considering the abundance and ubiquity of bacteriophages on Earth, the design of procedures for the removal of bacteriophages from bacterial cultures is crucial in diverse laboratory and industrial applications to preserve the integrity of the cultures and ensure accurate experimental results or product quality. Here, we have fine-tuned a protocol to eliminate the bacteriophages from infected Salmonella enterica cultures, using a strategy based on the use of lipopolysaccharides (LPS) located in the outer membrane of Gram-negative bacteria. Bacterial LPS plays an important role in host recognition by phages, and we make use of this property to design an effective procedure for the removal of phages, which use LPS as a receptor, in Salmonella bacterial cultures.

Author Spotlight: Efficiently Eliminating Bacteriophages from Infected Salmonella Cultures Using Lipopolysaccharides

Bacteriophages, ubiquitous and diverse on Earth, infect and replicate within bacterial hosts, playing a crucial role in microbial ecosystems. Despite their importance, their presence may disrupt industrial processes. We have developed a method using bacterial lipopolysaccharides to eliminate bacteriophages from Salmonella cultures.

Bacteriophages, or simply phages, play a vital role in microbial environments, impacting bacterial populations and shaping their evolution and ...

Biology

TIRF Microscopy-Based Visualization of Phagosome Formation and Closure

Source: Marie-Ana&#239;s, F., et al. <a href="https://app.jove.com/v/54470">&#34;Phagosome Closure Assay&#34; to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy (TIRFM).</a> J. Vis. Exp. (2016).
This video demonstrates a high-resolution total internal reflection fluorescence (TIRF) microscopy technique for real-time visualization of phagosome formation and closure during macrophage-mediated phagocytosis of IgG-opsonized red blood cells (RBCs) attached to the surface of a glass bottom dish. The macrophages extend pseudopodia around the RBCs and engulf them inside the phagosomes, detaching them from the glass surface.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
Note: The plasmid used ...

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Immunology

Immunodiagnostics

Imaging and Visualization Techniques

An Assay to Quantify Cytosolic and Vacuolar Salmonella in Infected Primary Macrophages

Source: Meunier, E., et al. <a href="https://app.jove.com/v/52960">Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization.</a> J. Vis. Exp. (2015).
This video demonstrates an assay to quantify cytosolic and vacuolar Salmonella typhimurium inside bone marrow-derived macrophages using differential permeabilization. The macrophages are infected with a fluorescent protein-expressing Salmonella, which is internalized inside a specialized vacuolar structure called Salmonella-containing vacuole or SCV. A subset of bacteria escape the SCV and enter the cytosol. The cholesterol-rich plasma membrane of macrophages is permeabilized using digitonin, leaving the SCV intact. Upon labeling the cytosolic bacteria with a fluorescently tagged antibody, the percentage of vacuolar and cytosolic bacteria is quantified using fluorescence-activated cell sorting (FACS).

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Summary

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Chapters in this video

A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

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

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

"Phagosome Closure Assay" to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy (TIRFM)

Characterizing Salmonella Typhimurium-induced Septic Peritonitis in Mice

Bacteriophage Removal from Infected Salmonella Cultures

TIRF Microscopy-Based Visualization of Phagosome Formation and Closure

An Assay to Quantify Cytosolic and Vacuolar Salmonella in Infected Primary Macrophages