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 enriched in bacteria and endosome/lysosome markers and devoid of cytosolic contamination.
This method presents several advantages when compared to the traditional phagosome isolation method that employs sucrose gradient separation. It eliminates the time-consuming multiple centrifugation steps, allowing the researcher to handle more samples in less time and obtain purer phagosome samples by easily increasing the number of washing steps. The use of this protocol eliminates the need for specialized and sensitive equipment, such as ultracentrifugation. High speed centrifugation could alter the characteristics of vesicles and reduce the final yield22, which is avoided in this protocol. We have also demonstrated that the coating of S. typhimurium with biotin and streptavidin-conjugated magnetic beads does not alter the phagocytic capacity of macrophages. Moreover, the induction of IL-6 by macrophages infected with labeled S. typhimurium was unaltered compared to macrophages infected with unlabeled bacteria. Therefore, coating of S. Typhimurium does not induce significant changes in their pathogenicity. To our knowledge, coating of S. typhimurium does not represent an impediment for the use of the isolated phagosomes for any of the common laboratory analysis techniques.
S. typhimurium-containing phagosomes isolated by this method are enriched in bacteria and present typical endosomal and lysosomal markers, such as the v-ATPase subunits V0 and V1 and the cleaved mature protease cathepsin D, which can be detected only in the isolated phagosome fractions. Additionally, we found mitochondria and ER markers in isolated S. typhimurium-containing phagosomes. Interaction of phagosomes with the ER has been widely studied although the amount of proteins from the ER present in pathogen-containing phagosomes may vary with the pathogen studied and the cell type. For example, phagosomes containing Brucella abortus isolated from nonprofessional phagocytes are rich in ER markers23 but not the ones isolated from macrophages24. Calnexin has been previously detected in S. Typhimurium-containing phagosomes isolated by sucrose gradient18. Close interaction of inert bead-containing phagosomes and mitochondria has also been previously reported25, explaining the presence of Tomm20 in isolated phagosomes. Mitochondrial proteins have been found in isolated phagosomes containing L. pneumophila26 or Mycobacterium27. However, the detection of mitochondria components in preparations of isolated pathogen-containing phagosomes by using the sucrose gradient centrifugation method has often been believed to be due to the similar density of this organelle and the pathogen-containing phagosome and, therefore, unspecific28. Our method avoids such a contamination issue and provides more reliable information suggesting that mitochondrial membranes might mingle with the phagosomal membranes. In summary, it will be difficult to avoid ER and mitochondrial markers in pathogen-containing isolated phagosomes preparations, depending on the host cell type and the pathogen. The recommended method for the verification of the purity of the isolated phagosomes is to use tagged-bacteria and specific tag antibodies for the detection of bacteria by Western Blot. We also show that this isolation method can be adapted for other microorganisms with available surface amine groups that can be biotinylated. As an example, we have successfully applied our protocol to isolate S. aureus-containing phagosomes. Phagosomes containing S. aureus isolated using this protocol are enriched in lysosome and endosome markers such as v-ATPase (V0 subunit) and Rab7, respectively, but do not present cytosolic markers such as GAPDH.
Endosomal/phagosomal vesicles provide unique environments for the degradation of pathogens, but preserve the antigens for signaling the adaptive immune system. Although, phagosomes and the maturation process have been extensively investigated, the microenvironment in the phagosome upon hosting various pathogens remains elusive. It is also unclear how metabolic changes in cells alter phagosome maturation and its content. Therefore, the protocol detailed here gives more opportunities to study phagosome biogenesis and its function in the context of various pathologies.

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>MgCl2</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&#181;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 incubated with streptavidin-conjugated magnetic beads. After 30 min incubation at 37 &#176;C, the cells were washed with warm PBS and fixed with 4% formaldehyde for 20 min at RT. Fixation was stopped by extensive washing with PBS. The cells were then permeabilized with 3% Triton in PBS for 5 min at RT. Infected BMDMs were then incubated with Cy5-Streptavidin for 20 min at 4 &#176;C. After extensive washing, the samples were prepared for analysis by confocal microscopy (Figure 1). Not biotinylated mCherry-S. Typhimurium was used as negative control. Fluorescent signal from Cy5-Streptavidin was observed exclusively in biotinylated mCherry-S. typhimurium, confirming that biotinylation of the bacteria was successful. Microscopical analysis was carried out on a confocal microscope using &#34;60X O2 NA:1.40&#34; objective. Excitation wavelengths for the fluorochromes were 405 nm (DAPI), 559 nm (mCherry), and 635 nm (Cy5), and PMT voltage was set at 574v, 565v, and 443v, respectively.
Since the immune response triggered in macrophages by S. typhimurium is largely dependent on the macrophage surface receptor activation by bacterial ligands, we next tested if the coating of S. typhimurium with biotin and streptavidin-conjugated magnetic beads could alter the innate immune response in infected BMDMs. We first assessed whether coating of S. typhimurium could alter the phagocytic capacity of BMDMs by confocal microscopy. BMDMs were infected as described in this protocol with mCherry-S. Typhimurium coated with biotin and streptavidin-conjugated magnetic beads or with uncoated mCherry-S. typhimurium. After 30 min incubation at 37 &#176;C, the cells were washed with warm PBS and fixed with 4% formaldehyde for 20 min at RT. After extensive washing with PBS, the samples were prepared for analysis by confocal microscopy. As shown in Figure 2, macrophages phagocytized coated and uncoated mCherry-S. typhimurium equally.
Next, we analyzed the inflammatory response triggered by S. typhimurium coated with biotin and streptavidin-conjugated magnetic beads (Figure 3). Supernatants from the BMDMs infected with coated and uncoated S. typhimurium were collected after 24 h of infection for enzyme-linked immunosorbent assay (ELISA) of secreted interleukin-6 (IL-6). The coating of S. typhimurium with biotin and streptavidin-conjugated magnetic beads did not significantly alter the inflammatory response of macrophages.
To assess the purity of the bacteria-containing phagosomes obtained by employing this protocol we analyzed isolated mCherry-S. typhimurium-containing phagosomes by immunoblotting. Using specific antibodies against the mCherry tag, we found significant enrichment of mCherry-expressing S. typhimurium in isolated phagosomes in comparison to the cytosolic fraction obtained from the same sample (Figure 4). Isolated phagosomes containing mCherry- S. typhimurium that was not previously coated with biotin were used as a negative control. These results demonstrate that this protocol allows for the purification of phagosomes that are highly enriched in bacteria.
We next performed immunoblot analysis of endosome and lysosome markers in isolated phagosomes containing S. typhimurium (Figure 5). Most of the endosome and lysosome markers were clearly observed in phagosomes isolated at 4 h post-infection in comparison to 30 min. The enrichment of the endosomal markers Rab5 and Rab7, as well as the lysosome markers v-ATPase (both V0 and V1 subunits) and cathepsin D, were observed. Interestingly, only the cleaved form of cathepsin D in isolated phagosomes at 4 h post-infection was observed. The cleavage of pro-cathepsin D (48 kDa) to produce the short active form (33 kDa) only occurs in the phagosomes but not in the cytosol, demonstrating the specificity of this protocol for phagosome isolation.
Since markers from organelles such as mitochondria or endoplasmic reticulum (ER) are often detected in preparations of bacteria-containing isolated phagosomes, the S. typhimurium-containing isolated phagosomes were probed with specific antibodies against the mitochondrial transporter Tomm20 and the ER protein calnexin (Figure 6). We also tested them with an antibody against the glycolysis enzyme GAPDH to assess cytosolic contamination in the isolated phagosomes. We found both mitochondria and ER markers in S. typhimurium-isolated phagosomes but no cytosolic contamination.
To study the versatility of the protocol for isolation of bacteria-containing phagosomes, the biotin and streptavidin-conjugated magnetic beads coating procedure was applied to the Gram-positive bacterium S. aureus. By employing the same fixation and staining protocol as described for mCherry-S. typhimurium (Figure 1) we confirmed successful biotinylation of green fluorescent protein (GFP) expressing-S. aureus (Figure 7). Moreover, we detected enrichment of the endosome marker Rab7 and the lysosome markers v-ATPase (V0 subunit) and cathepsin D by immunoblot analysis of isolated GFP-S. aureus-containing phagosomes (Figure 8). The cleavage of cathepsin D into its mature form was only detectable after 4 h of infection in the isolated phagosome samples but not in the cytosolic fractions. Isolated S. aureus-containing phagosomes were free of cytosolic contamination as shown by the immunodetection of GAPDH.
In summary, we have demonstrated that our phagosome isolation protocol enables the isolation of highly enriched bacteria-containing phagosomes, free of cytosolic contamination. The isolated phagosome preparations can be used for the analysis of endosome and lysosome markers to study phagosome maturation. Moreover, it was shown that this protocol can be used for both Gram-negative and Gram-positive bacteria and that the labeling procedure does not alter the immune response of macrophages.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56514/56514fig1.jpg" /> 
Figure 1: Fluorescence microscopy analysis of biotinylated S. typhimurium using Cy5-Streptavidin. BMDMs were infected for 30 min with mCherry-S. typhimurium that was biotinylated (&#34;Biotinylated S. T.&#34;), as described in this protocol. Not biotinylated mCherry-S. typhimurium (&#34;Not biotinylated S. T.&#34;) was used as a negative control. After fixation and permeabilization of the cells, samples were incubated with Cy5-Streptavidin for 20 min at 4 &#176;C. Fluorescence signal from the nucleus (DAPI), mCherry-S. typhimurium, and Cy5-Streptavidin were analyzed using confocal microscopy. Signal from the Cy5-Streptavidin could only be detected in the bacteria previously labeled with biotin, thus confirming efficient biotinylation. Scale bar = 10 &#181;m. <a href="https://cloudflare.jove.com/files/ftp_upload/56514/56514fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56514/56514fig2.jpg" /> 
Figure 2: Microscopical analysis of BMDMs infected with mCherry-S. typhimurium coated with biotin and streptavidin-conjugated magnetic beads. BMDMs were infected with mCherry-S.typhimurium labeled with biotin and streptavidin-conjugated magnetic beads as described in this protocol. After allowing the macrophages to phagocytize the bacteria for 30 min, cells were fixed with 4% formaldehyde for 20 min at RT. The coated bacteria phagocytized by macrophages were then analyzed by confocal microscopy. Analysis showed that the intake of coated bacteria (&#34;Coated S.T.&#34;) is comparable to the intake of unlabeled mCherry bacteria (&#34;Not coated S.T.&#34;), demonstrating that labeling with biotin and streptavidin-conjugated magnetic beads does not alter phagocytosis of S. typhimurium by macrophages. Nuclei were stained with DAPI for microscopy visualization. Data are shown as mean &#177; S.E.M., and the statistical significance calculated using student t-test is represented as * = p &#60;0.05; ** = p &#60;0.01; *** = p &#60;0.001. Scale bar = 40 &#181;m. <a href="https://cloudflare.jove.com/files/ftp_upload/56514/56514fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56514/56514fig3.jpg" /> 
Figure 3: IL-6 secretion by BMDMs infected with biotin-and-streptavidin-conjugated magnetic beads, labeled S. typhimurium compared to unlabeled S. typhimurium. BMDMs were infected with S. typhimurium coated with biotin and streptavidin-conjugated magnetic beads as described in this protocol. After 24 h of infection, supernatants were collected for further analysis of IL-6 secretion by ELISA. Supernatants form BMDMs infected with uncoated S. typhimurium were used as a control. The induction of IL-6 secretion by coated S. typhimurium was not significantly different compared to that of uncoated S. typhimurium. Data are shown as mean &#177; S.E.M., and the statistical significance calculated using student t-test is represented as * = p &#60;0.05; ** = p &#60;0.01; *** = p &#60;0.001. <a href="https://cloudflare.jove.com/files/ftp_upload/56514/56514fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56514/56514fig4.jpg" /> 
Figure 4: Bacterial enrichment in isolated phagosomes. Enrichment of mCherry-tagged S. typhimurium in isolated phagosomes collected after 30 min and 4 h of infection was compared to the cytosolic fraction. &#946;-actin was used as a loading control. The negative control refers to phagosomes isolated using uncoated S. typhimurium at 30 min post-infection. As expected, the final output of phagosome isolation using unlabeled S. typhimurium does not present significant amounts of mCherry or &#946;-actin, demonstrating the specificity of the protocol. 20 &#181;g of protein was loaded per sample. <a href="https://cloudflare.jove.com/files/ftp_upload/56514/56514fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56514/56514fig5.jpg" /> 
Figure 5: Analysis of endosome and lysosome markers in isolated S. typhimurium-containing phagosomes. The enrichment of the endosome markers Rab5 and Rab7, and the lysosome markers v-ATPase (V0 and V1 subunits) and cathepsin D was analyzed in isolated S. typhimurium-containing phagosomes extracted after 30 min and 4 h of infection. &#946;-actin was used as a loading control. 20 &#181;g of protein was loaded per sample. <a href="https://cloudflare.jove.com/files/ftp_upload/56514/56514fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56514/56514fig6.jpg" /> 
Figure 6: Analysis of mitochondria, ER, and cytosolic markers in isolated S. typhimurium-containing phagosomes. The cytosolic marker GAPDH, the ER marker calnexin, and the mitochondrial marker Tomm20 in S. typhimurium phagosomes isolated at 30 min and 4 h post-infection were analyzed by immunoblotting and compared to the corresponding cytosolic fractions. 20 &#181;g of protein were loaded per sample. <a href="https://cloudflare.jove.com/files/ftp_upload/56514/56514fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/56514/56514fig7.jpg" /> 
Figure 7: Microscopical analysis of GFP-S. aureus infected macrophages. BMDMs were infected with S. aureus expressing green fluorescent protein (GFP) labeled with biotin as described in methodology for labeling S. typhimurium. After allowing macrophages to phagocytize the bacteria for 30 min, the cells were fixed. Uncoated S. aureus (&#34;Not biotinylated S. A.&#34;) were used as negative control. After fixation and permeabilization of the cells, samples were incubated with Cy5-Streptavidin for 20 min at 4 &#176;C. The fluorescent signal from the nucleus (DAPI), GFP-S. aureus, and Cy5-Streptavidin were analyzed by confocal microscopy. The signal from Cy5-Streptavidin could only be seen in the bacteria previously labeled with biotin, confirming efficient biotinylation. Scale bars = 10 &#181;m. <a href="https://cloudflare.jove.com/files/ftp_upload/56514/56514fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/56514/56514fig8.jpg" /> 
Figure 8: Analysis of endosome and lysosome markers in isolated S. aureus-containing phagosomes. S. aureus-containing phagosomes isolated at 30 min, 2 h, and 4 h post-infection, were analyzed by immunoblotting for phagosomal markers Rab5, Rab7, and v-ATPase compared to the corresponding cytosolic fractions. 20 &#181;g of protein were loaded per sample. Samples were also tested with a specific antibody against GAPDH, demonstrating that isolated S. aureus-containing phagosomes are free of cytosolic contamination. <a href="https://cloudflare.jove.com/files/ftp_upload/56514/56514fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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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Immunology and Infection

10.5K 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

Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and food antigens while concomitantly remaining poised to mount inflammatory responses toward invasive pathogens1,2. Antigen presenting cells, particularly DCs and macrophages, play critical roles in maintaining intestinal immune homeostasis via their ability to sense and appropriately respond to the microbiota3-14. Efficient isolation of intestinal DCs and macrophages is a critical step in characterizing the phenotype and function of these cells. While many effective methods of isolating intestinal immune cells, including DCs and macrophages, have been described6,10,15-24, many rely upon long digestions times that may negatively influence cell surface antigen expression, cell viability, and/or cell yield. Here, we detail a methodology for the rapid isolation of large numbers of viable, intestinal DCs and macrophages. Phenotypic characterization of intestinal DCs and macrophages is carried out by directly staining isolated intestinal cells with specific fluorescence-labeled monoclonal antibodies for multi-color flow cytometric analysis. Furthermore, highly pure DC and macrophage populations are isolated for functional studies utilizing CD11c and CD11b magnetic-activated cell sorting beads followed by cell sorting.

Here, we detail a methodology for the rapid isolation of mouse intestinal dendritic cells (DCs) and macrophages. Phenotypic characterization of intestinal DCs and macrophages is performed using multi-color flow cytometric analysis while magnetic bead enrichment followed by cell sorting is used to yield highly pure populations for functional studies.

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and ...

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote its motility and dissemination. New work has shown that proteins involved in actin-based motility are also linked to autophagy, an intracellular degradation process crucial for cell autonomous immunity. Strikingly, host cells may prevent actin-based motility of S. flexneri by compartmentalizing bacteria inside &#8216;septin cages&#8217; and targeting them to autophagy. These observations indicate that a more complete understanding of septins, a family of filamentous GTP-binding proteins, will provide new insights into the process of autophagy. This report describes protocols to monitor autophagy-cytoskeleton interactions caused by S. flexneri in vitro using tissue culture cells and in vivo using zebrafish larvae. These protocols enable investigation of intracellular mechanisms that control bacterial dissemination at the molecular, cellular, and whole organism level.

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

To counteract pathogen dissemination, host cells reorganize their cytoskeleton to compartmentalize bacteria and induce autophagy. Using Shigella infection of tissue culture cells, host and pathogen determinants underlying this process are identified and characterized. Using zebrafish models of Shigella infection, the role of discovered molecules and mechanisms are investigated in vivo.

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote ...

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 ...

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 ...

Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry

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

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

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

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.

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.

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

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

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

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

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

Isolation and 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 ...

Chronic Salmonella Infection Induced Intestinal Fibrosis

Tissue fibrosis characterized by the pathological accumulation of extracellular matrix such as collagen is the outcome of persistent inflammation and dysregulated repair. In inflammatory bowel disease (IBD), fibrosis leads to recurrent stricture formations for which there is no effective therapy other than surgical resection. Due to its late onset, the processes that drive fibrosis is less studied and largely unknown. Therefore, fibrotic complications represent a major challenge in IBD. In this protocol, a robust in vivo model of intestinal fibrosis is described where streptomycin pre-treatment of C57Bl/6 mice followed by oral gavage with vaccine grade Salmonella Typhimurium &#916;AroA mutant leads to persistent pathogen colonization and fibrosis of the cecum. Methodologies for preparing S. Typhimurium &#916;AroA for inoculation, quantifying pathogen loads in the cecum and spleen, and evaluating collagen deposition in intestinal tissues are explained. This experimental disease model is useful for examining host factors that either enhance or exacerbate CD-like intestinal fibrosis.

This protocol describes a mouse model of Salmonella driven intestinal fibrosis that resembles key pathological hallmarks of Crohn&#8217;s disease including transmural inflammation and fibrosis. This method can be used to evaluate host factors that alter fibrotic outcomes using mutant mice maintained on a C57Bl/6 genetic background.

Tissue fibrosis characterized by the pathological accumulation of extracellular matrix such as collagen is the outcome of persistent inflammation and ...

Production and Characterization of Human Macrophages from Pluripotent Stem Cells

Macrophages are present in most vertebrate tissues and comprise widely dispersed and heterogeneous cell populations with different functions. They are key players in health and disease, acting as phagocytes during immune defense and mediating trophic, maintenance, and repair functions. Although it has been possible to study some of the molecular processes involved in human macrophage function, it has proved difficult to apply genetic engineering techniques to primary human macrophages. This has significantly hampered our ability to interrogate the complex genetic pathways involved in macrophage biology and to generate models for specific disease states. An off-the-shelf source of human macrophages that is amenable to the vast arsenal of genetic manipulation techniques would, therefore, provide a valuable tool in this field. We present an optimized protocol that allows for the generation of macrophages from human induced pluripotent stem cells (iPSCs) in vitro. These iPSC-derived macrophages (iPSC-DMs) express human macrophage cell surface markers, including CD45, 25F9, CD163, and CD169, and our live-cell imaging functional assay demonstrates that they exhibit robust phagocytic activity. Cultured iPSC-DMs can be activated to different&#160;macrophage states that display altered gene expression and phagocytic activity by the addition of LPS and IFNg, IL4, or IL10. Thus, this system provides a platform to generate human macrophages carrying genetic alterations that model specific human disease and a source of cells for drug screening or cell therapy to treat these diseases.

This protocol describes the robust generation of macrophages from human induced pluripotent stem cells, and methods for their subsequent characterization. Cell surface marker expression, gene expression, and functional assays are used to assess the phenotype and function of these iPSC-derived macrophages.

Macrophages are present in most vertebrate tissues and comprise widely dispersed and heterogeneous cell populations with different functions. They are key ...