This work was supported by a 'Grant in Aid of Research' from the Crohn's and Colitis Foundation of Canada to LMS, who is supported by a Canadian Association of Gastroenterology/Canadian Association of Health Research/Crohn's and Colitis Foundation of Canada New Investigator Award.

1. Depleting Macrophages Using Clodronate-containing Liposomes
<ol>
 <li>Liposomes are stored at 4 &deg;C. Two hours prior to injection, remove clodronate-containing liposomes, PBS-containing liposomes, or sterile PBS (injection control) from the refrigerator to allow them to acclimate to room temperature (18 &deg;C).</li>
 <li>Invert the tubes containing liposomes 8-10 times to ensure an even distribution before loading 200 &mu;l into a 1 ml syringe. Attach a 26 gauge needle to the top of the syringe.</li>
 <li>Using your left hand, scruff the mouse just below the ears holding enough skin to immobilize its head and limbs.</li>
 <li>Tilt the mouse so its head is slightly towards the ground so internal organs move away from the injection site, which is located in the lower right quadrant of the abdomen.</li>
 <li>Roll the syringe between your palms and invert it 6 times to distribute the liposome solution evenly prior to injection.</li>
 <li>Insert the needle in the lower right side of the mouse abdomen at a 30-40 degree angle. Inject 200 &mu;l of liposome solution or PBS.</li>
 <li>During an induced inflammation model, like DSS-induced colitis, inject mice 4 days prior to onset of experimental inflammation to ensure depletion of resident macrophages and every 2 days during the protocol to deplete new infiltrating macrophages.</li>
 <li>At the end of the experiment, along with planned experimental analyses, remove tissues from the site of interest and fix with paraformaldehyde to confirm macrophage depletion by immunohistochemical staining.</li>
</ol>
2. Derivation of Polarized Macrophages from Bone Marrow Aspirates
<ol>
 <li>Bone marrow from mice between 8 and 12 weeks of age provides the highest yield of bone marrow-derived macrophages. Euthanize the mouse, lay it on its back, and pin down its paws, stretching its limbs as far as possible.</li>
 <li>Working in a sterile hood, remove femurs and tibias, cutting away as much muscle as possible.</li>
 <li>Flush marrow from bones using IMDM with 10% FCS loaded into a 5 ml syringe fitted with a 26 gauge needle.</li>
 <li>Dilute bone marrow aspirates to 40 ml in IMDM with 10% FCS, place cells in a 75 cm2 tissue culture flask, and incubate at 37 &deg;C, 5% CO2 for 4 hours. This step removes adherent mesenchymal cells or mature macrophages from hematopoietic progenitors.</li>
 <li>Transfer culture supernatant containing non-adherent progenitors to a 50 ml conical Falcon tube and centrifuge for 5 min at 1200 rpm.</li>
 <li>Resuspend cell pellet in 5 ml IMDM, 10% FCS and count nucleated cells by diluting cell suspension 1 in 20 in acetic acid. This procedure lyses all cells including red blood cells and the remaining nuclei can be counted on a hemocytometer.</li>
 <li>Resuspend cells at a concentration of 0.5&times;106 cells/ml in complete medium; IMDM, 10% FCS, 150 &mu;M monothioglycerol (MTG), and 10 ng/ml of either MCSF, GM-CSF, or IL-3. MCSF-derived macrophages are classically activated macrophages whereas GM-CSF- or IL-3-derived macrophages are alternatively activated.</li>
 <li>Replace medium at day 4, spinning down non-adherent cells and returning them to the flask, and at day 7, discarding non-adherent cells.</li>
 <li>In addition, IFN&gamma; (10 ng/ml) or IL-4 (10 ng/ml) can be added to flasks containing MCSF-derived cells at day 7 to skew macrophages to a classically activated or an alternatively activated phenotype, respectively. Incubate cells for 3 more days.</li>
 <li>At day 10, remove media and lift cells off of the tissue culture flask using Cell Dissociation Buffer (Gibco-BRL). Place 5 ml of buffer on cells for 5 min at room temperature (18 &deg;C) and then bang the side of the flask with the heel of your palm firmly several times. Ensure that cells have lifted off of the flask by examining the flask under a microscope. Pipet resuspended cells into a 15 ml conical Falcon tube and wash the flask with an additional 10 ml of IMDM, 10% FBS. Pool and spin down the cells for 5 min at 300&times;g.</li>
 <li>Count viable cells using a hemocytometer and resuspend at a concentration of 107 cells/ml in sterile PBS pH7.4. Macrophages are ready for injection into mice.</li>
 <li>Reserve 1.0&times;106 macrophages for assessment of macrophage phenotype. Classically activated macrophages stimulated with lipopolysaccharide secrete high levels of nitric oxide and IL-12p70 and low levels of IL-10 compared to alternatively activated macrophages. Alternatively activated macrophages constitutively express YM1 and arginase I (ArgI), which can be detected by Western immunoblotting.</li>
</ol>
3. Tranferring Macrophages into Mice
<ol>
 <li>Resuspend macrophages in sterile PBS at a concentration of 107 cells/ml. This allows for an intravenous injection of 106 macrophages in a total volume of 100 &mu;l, which is a volume that can be safely and comfortably injected into mice by the tail vein.</li>
 <li>Warm mice using a heating pad or heat lap for 5-10 min to dilate the tail vein. Monitor the mouse at all times for signs of hyperthermia.</li>
 <li>Transfer the mouse to a restraining device that permits access to the tail veins.</li>
 <li>Rotate the tail 90&deg; so that the vein is facing upward. Veins run laterally down both sides of the tail and either vein can be used.</li>
 <li>Clean the injection site with an alcohol swab and slowly inject 100 &mu;l ( 106 macrophages) using a 1 ml syringe with a 26 or 28 gauge needle. Insert the needle with the bevel side facing up and at a slight angle, almost parallel to the vein.</li>
 <li>If the needle is inserted properly no resistance should be felt and the vein should become clear after injection. If the needle is not in the vein, the tail will bulge. If this happens, remove the needle and try again proximal to the last attempt or in the other vein. A new needle must be used for each injection as the tail is tough and the needles become dull quickly during tail vein injection.</li>
 <li>Following injection, remove the needle, apply gentle pressure to the injection site until the bleeding stops, and monitor the mouse for an additional 5min to ensure that bleeding has stopped.</li>
 <li>Repeat macrophage injections every 4 days to ensure a continuous delivery of polarized macrophages during the experimental procedure.</li>
 <li>At the end of the experiment, along with experimental analyses, remove tissues from the site of interest and fix with paraformaldehyde or formalin to confirm effective delivery of polarized macrophages by immunohistochemical staining.</li>
</ol>
4. Representative Results
The number and phenotype of resident macrophages in any given tissue depends on the tissue being examined and will change during infection or inflammation. Similarly, the ability of clodronate-containing liposomes to deplete macrophages from a tissue will depend on the route of administration and effective delivery to the target tissue. Intraperitoneal injection of clodronate-containing liposomes results in a decrease in F4/80+ macrophages in the mouse colon during DSS-induced colitis evident by immunohistochemical staining for the macrophage marker, F4/80 (Figure 1A). Histological staining can also be used to determine quantitative differences in macrophage number and phenotype in tissue sections. Figure 1B shows that an average of 55% of macrophages is depleted by clodronate-containing liposomes compared to no depletion detected in mouse colons when mice are injected with PBS alone as an injection control. Each value is determined by counting F4/80 and ArgI stained positive cells in serial tissue sections from 6 mice at 3 points, in 3 sections per mouse with counting being performed by two individuals blinded to experimental condition. These results demonstrate that intraperitoneal injections of clodronate liposomes deplete colonic macrophages in mice.
Derivation of macrophages from bone marrow in the presence of different growth factors yields macrophages that are classically activated (MCSF-derived or MCSF-derived and treated with IFN&gamma;) or alternatively activated (GM-CSF or IL-3-derived or MCSF-derived and treated with IL-4). Flushing bone marrow from 2 femurs and 2 tibias from an 8 week old mouse typically yields 6&times;107 nucleated cells per mouse and yields 8&times;106 MCSF-derived macrophages, 10&times;106 GM-CSF-derived macrophages, or 12&times;106 IL-3-derived macrophages. Figure 2A shows nitrite and the IL-12p70/IL-10 ratio in macrophage supernatants treated with lipopolysaccharide for 24 hours. Figure 2B shows a typical Western blot analysis of 0.5&times;106 macrophages from MCSF +/- IL-4 derivation conditions probed with antibodies for alternatively activated macrophage markers, ArgI, YM1, or with GAPDH as a loading control. These data demonstrate that bone marrow-derived macrophages can be skewed to a polarized phenotype during derivation.
Effective transfer of ex vivo derived polarized macrophages to a tissue is dependent on the route of administration and access to the tissue. During DSS-induced colitis, macrophages injected intravenously travel to the site of inflammation and alternatively activated macrophages reduce disease severity. Figure 3A shows the total number of macrophages and the number of ArgI+ (alternatively activated) macrophages counted in histological sections from mice injected with polarized bone marrow-derived macrophages. The impact of transfer of polarized macrophages to the colon during DSS-induced colitis is shown by disease activity index, an additive score based on weight loss, rectal bleeding, and decreased stool consistency (Figure 3B). These results demonstrate that ex vivo derived, adoptively transferred macrophages can traffic to the colon and impact induced inflammation. M1 macrophages do not impact inflammation whereas M2 macrophages significantly reduce disease activity index.
<img src="/files/ftp_upload/4105/4105fig1.jpg" alt="Figure 1"/> 
Figure 1. Clodronate-containing liposomes effectively deplete F4/80+ and F480+ArgI+ cells in mouse colons in vivo. An F2 generation of wild type mice on a mixed C57BL/6 &times;129Sv background were given 5% DSS for 7 days and injected IP with PBS (Control) or clodronate-containing liposomes (Clodronate) 4 days prior to DSS treatment and on days 0, 2, 4, and 6 during DSS treatment. (A) Colons were removed, fixed, and sections were stained by immunohistochemistry for mature macrophages (F4/80) and the M2 macrophage marker, ArgI. (B) Quantitation of F4/80+ and ArgI+ macrophages. Positively stained cells were counted by two individuals blinded to experimental condition at 40&times; magnification in 6 fields from 3 sections/mouse and from 6 mice/group. *P&lt;0.01.
<img src="/files/ftp_upload/4105/4105fig2.jpg" alt="Figure 2"/> 
Figure 2. MCSF-derived macrophages express a classically activated phenotype and MCSF-derived macrophages treated with IL-4 express an alternatively activated macrophage phenotype. (A) Consistent with a classically activated phenotype, MCSF-derived macrophages produce higher levels of nitric oxide in response to lipopolysaccharide and can be measured by the Griess assay, which detects its downstream metabolite, nitrite. They also produce a higher IL-12p70/IL-10 ratio, which can be measured by ELISA. *P&lt;0.01 for n=3. (B) Consistent with an alternatively activated phenotype, MCSF-derived macrophages treated with IL-4 constitutively express YM1 and ArgI, which can be detected by Western immunoblotting. Data are representative of 3 independent experiments with similar results.
<img src="/files/ftp_upload/4105/4105fig3.jpg" alt="Figure 3"/> 
Figure 3. Intravenous injected, ex vivo-derived alternatively activated macrophages traffic to the colon and reduce intestinal inflammation. An F2 generation of wild type mice on a mixed C57BL/6 &times;129Sv background were given 5% DSS in their drinking water for six days. M1 and M2 macrophages were injected IV on Days 0 and 4 during DSS treatment. (A) Colons were removed, fixed, and sections were stained by immunohistochemistry for mature macrophages (F4/80) and an M2 macrophage marker (ArgI). Quantitation of macrophages for control PBS injected mice (C), mice injected with M1 macrophages (+M1) and mice injected with M2 macrophages (+M2) were performed by counting positively stained cells at 40&times; magnification in 6 fields from 3 sections/mouse and from 6 mice/group. Counting was performed by two individuals blinded to experimental condition. (B) Disease activity was monitored and scored daily during treatment and is an additive score based on weight loss, rectal bleeding, and decreased stool consistency. *P&lt;0.05 for n= 6 mice in 2 independent experiments.

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	<li>Gordon, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+activation+of+macrophages.">Alternative activation of macrophages.</a> Nat. Rev. Immunol. 3, 23-35 (2003).</li><li>Rescigno, M., Lopatin, U., Chieppa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+among+dendritic+cells,+macrophages,+and+epithelial+cells+in+the+gut:+implications+for+immune+tolerance.">Interactions among dendritic cells, macrophages, and epithelial cells in the gut: implications for immune tolerance.</a> Curr. Opin. Immunol. 20, 669-675 (2008).</li><li>Heinsbroek, S. 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No conflicts of interest declared.

Macrophages are phagocytic cells that play an important role in the immune system. They are responsible for initiating the innate immune response and directing the acquired immune response. Typically, classically activated macrophages are activated by IFN&gamma; or LPS and are responsible for eliminating pathogens and mounting an inflammatory response1. Conversely, alternatively activated macrophages are activated by IL-4 or IL-13 and play a role in debris scavenging and tissue remodeling during the...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>Clodronate-containing liposomes</td>
 <td><a href="http://Clodronateliposomes.org" target="_blank">Clodronateliposomes.org</a></td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>PBS-containing liposomes</td>
 <td><a href="http://Clodronateliposomes.org" target="_blank">Clodronateliposomes.org</a></td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Sterile PBS pH7.4 </td>
 <td>Invitrogen</td>
 <td>14190</td>
 </tr>
 <tr>
 <td>IMDM</td>
 <td>Invitrogen</td>
 <td>12440</td>
 </tr>
 <tr>
 <td>Fetal Calf Serum (FCS)</td>
 <td>Invitrogen</td>
 <td>12483 </td>
 </tr>
 <tr>
 <td>acetic acid</td>
 <td>BDH Aristar</td>
 <td>BDH3092-500MLP </td>
 </tr>
 <tr>
 <td>Monothioglycerol (MTG)</td>
 <td>Sigma</td>
 <td>96-275</td>
 </tr>
 <tr>
 <td>Recombinant murine MCSF</td>
 <td>Stemcell Technologies</td>
 <td>02951</td>
 </tr>
 <tr>
 <td>Recombinant murine GM-CSF</td>
 <td>Stemcell Technologies</td>
 <td>02935</td>
 </tr>
 <tr>
 <td>Recombinant murine IL-3</td>
 <td>Stemcell Technologies</td>
 <td>02903</td>
 </tr>
 <tr>
 <td>Recombinant murine IFN&gamma; </td>
 <td>Stemcell Technologies</td>
 <td>02746</td>
 </tr>
 <tr>
 <td>Recombinant murine IL-4</td>
 <td>Stemcell Technologies</td>
 <td>02714</td>
 </tr>
 <tr>
 <td>Cell Dissociation Buffer </td>
 <td>Invitrogen</td>
 <td>13150-016</td>
 </tr>
 <tr>
 <td>Rat anti-F4/80 Antibody</td>
 <td>AbD Serotec </td>
 <td>MCA497GA</td>
 </tr>
 <tr>
 <td>Mouse anti-ArgI Antibody</td>
 <td>BD Transduction Laboratories</td>
 <td>610708</td>
 </tr>
 </tbody>
</table>
Table 1. Specific reagents used in this protocol.

depletion and reconstitution of macrophages in mice

Macrophages are critical players in the innate immune response to infectious challenge or injury, initiating the innate immune response and directing the acquired immune response. Macrophage dysfunction can lead to an inability to mount an appropriate immune response and as such, has been implicated in many disease processes, including inflammatory bowel diseases. Macrophages display polarized phenotypes that are broadly divided into two categories. Classically activated macrophages, activated by stimulation with IFN&gamma; or LPS, play an essential role in response to bacterial challenge whereas alternatively activated macrophages, activated by IL-4 or IL-13, participate in debris scavenging and tissue remodeling and have been implicated in the resolution phase of inflammation. During an inflammatory response in vivo, macrophages are found amid a complex mixture of infiltrating immune cells and may participate by exacerbating or resolving inflammation. To define the role of macrophages in situ in a whole animal model, it is necessary to examine the effect of depleting macrophages from the complex environment. To ask questions about the role of macrophage phenotype in situ, phenotypically defined polarized macrophages can be derived ex vivo, from bone marrow aspirates and added back to mice, with or without prior depletion of macrophages. In the protocol presented here clodronate-containing liposomes, versus PBS injected controls, were used to deplete colonic macrophages during dextran sodium sulfate (DSS)-induced colitis in mice. In addition, polarized macrophages were derived ex vivo and transferred to mice by intravenous injection. A caveat to this approach is that clodronate-containing liposomes deplete all professional phagocytes, including both dendritic cells and macrophages so to ensure the effect observed by depletion is macrophage-specific, reconstitution of phenotype by adoptive transfer of macrophages is necessary. Systemic macrophage depletion in mice can also be achieved by backcrossing mice onto a CD11b-DTR background, which is an excellent complementary approach. The advantage of clodronate-containing liposome-mediated depletion is that it does not require the time and expense involved in backcrossing mice and it can be used in mice regardless of the background of the mice (C57BL/6, BALB/c, or mixed background).

Watch this Scientific Journal Video about Depletion and Reconstitution of Macrophages in Mice at JoVE.com

Depletion and Reconstitution of Macrophages in Mice

Macrophages play a central role in homeostasis and pathology in many tissues. The protocol presented here describes methods for depleting macrophages in vivo, deriving polarized macrophages from bone marrow aspirates, and adoptively transferring macrophages into mice. These techniques allow determination of the role that polarized macrophages play in health and disease.

Macrophages are critical players in the innate  immune response to infectious challenge or injury, initiating the innate immune  response and directing ...

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39.4K Views.  University of British Columbia. Macrophages play a central role in homeostasis and pathology in many tissues. The protocol presented here describes methods for depleting macrophages in vivo, deriving polarized macrophages from bone marrow aspirates, and adoptively transferring macrophages into mice. These techniques allow determination of the role that polarized macrophages play in health and disease.

Video: Depletion and Reconstitution of Macrophages in Mice

Depletion of Specific Cell Populations by Complement Depletion

The purification of immune cell populations is often required in order to study their unique functions. In particular, molecular approaches such as real-time PCR and microarray analysis require the isolation of cell populations with high purity. Commonly used purification strategies include fluorescent activated cell sorting (FACS), magnetic bead separation and complement depletion. Of the three strategies, complement depletion offers the advantages of being fast, inexpensive, gentle on the cells and a high cell yield. The complement system is composed of a large number of plasma proteins that when activated initiate a proteolytic cascade culminating in the formation of a membrane-attack complex that forms a pore on a cell surface resulting in cell death1. The classical pathway is activated by IgM and IgG antibodies and was first described as a mechanism for killing bacteria. With the generation of monoclonal antibodies (mAb), the complement cascade can be used to lyse any cell population in an antigen-specific manner. Depletion of cells by the complement cascade is achieved by the addition of complement fixing antigen-specific antibodies and rabbit complement to the starting cell population. The cells are incubated for one hour at 37&deg;C and the lysed cells are subsequently removed by two rounds of washing. MAb with a high efficiency for complement fixation typically deplete 95-100% of the targeted cell population. Depending on the purification strategy for the targeted cell population, complement depletion can be used for cell purification or for the enrichment of cell populations that then can be further purified by a subsequent method.

To effectively study the function of immune cell populations their purification is often required. Complement depletion is a fast and inexpensive technique for the isolation of immune cell populations with high purity.

The purification of immune cell populations is often required in order to study their unique functions.  In particular, molecular approaches such as ...

Study of Phagolysosome Biogenesis in Live Macrophages

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome maturation is the complex and tightly regulated process during which a nascent phagosome undergoes drastic transformation through well-orchestrated interactions with various cellular organelles and compartments in the cytoplasm. This process, which is essential for the physiological function of phagocytic cells by endowing phagosomes with their lytic and bactericidal properties, culminates in fusion of phagosomes with lysosomes and biogenesis of phagolysosomes which is considered to be the last and critical stage of maturation for phagosomes. In this report, we describe a live cell imaging based method for qualitative and quantitative analysis of the dynamic process of lysosome to phagosome content delivery, which is a hallmark of phagolysosome biogenesis. This approach uses IgG-coated microbeads as a model for phagocytosis and fluorophore-conjugated dextran molecules as a luminal lysosomal cargo probe, in order to follow the dynamic delivery of lysosmal content to the phagosomes in real time in live macrophages using time-lapse imaging and confocal laser scanning microscopy. Here we describe in detail the background, the preparation steps and the step-by-step experimental setup to enable easy and precise deployment of this method in other labs. Our described method is simple, robust, and most importantly, can be easily adapted to study phagosomal interactions and maturation in different systems and under various experimental settings such as use of various phagocytic cells types, loss-of-function experiments, different probes, and phagocytic particles.

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome ...

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 ...

Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied by adverse side effects such as a reduced ability to control infections or malignancies. Therefore, new approaches need to be developed that target only the disease mediating cells and leave the remaining immune system intact. Over the past decade a variety of cell based immunotherapy strategies to modulate T cell mediated immune responses have been developed. Most of these approaches rely on tolerance-inducing antigen presenting cells (APC). However, in addition to being technically difficult and cumbersome, such cell-based approaches are highly sensitive to cytotoxic T cell responses, which limits their therapeutic capacity. Here we present a protocol for the generation of non-cellular killer artificial antigen presenting cells (KaAPC), which allows for the depletion of pathologic T cells while leaving the remaining immune system untouched and functional. KaAPC is an alternative solution to cellular immunotherapy which has potential for treating autoimmune diseases and allograft rejections by regulating undesirable T cell responses in an antigen specific fashion.

Killer Artificial Antigen Presenting Cells KaAPC for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Guidelines are presented for the generation of killer artificial antigen presenting cells, KaAPC, an efficient tool for in vitro depletion of human antigen-specific T cells and an alternative solution to cellular immunotherapy for the treatment of T cell-mediated autoimmune diseases in an antigen-specific fashion without compromising the remaining immune system.

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied ...

Proteomic Profiling of Macrophages by 2D Electrophoresis

The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The key role of macrophages has been shown in various pathological disorders such as inflammatory, immunological, and infectious diseases. In this protocol, we use primary cultures of human monocyte-derived macrophages that can be differentiated into the M1 (pro-inflammatory) or the M2 (anti-inflammatory) phenotype. This in vitro model is reliable for studying the biological activities of M1 and M2 macrophages and also for a proteomic approach. Proteomic techniques are useful for comparing the phenotype and behaviour of M1 and M2 macrophages during host pathogenicity. 2D gel electrophoresis is a powerful proteomic technique for mapping large numbers of proteins or polypeptides simultaneously. We describe the protocol of 2D electrophoresis using fluorescent dyes, named 2D Differential Gel Electrophoresis (DIGE). The M1 and M2 macrophages proteins are labelled with cyanine dyes before separation by isoelectric focusing, according to their isoelectric point in the first dimension, and their molecular mass, in the second dimension. Separated protein or polypeptidic spots are then used to detect differences in protein or polypeptide expression levels. The proteomic approaches described here allows the investigation of the macrophage protein changes associated with various disorders like host pathogenicity or microbial toxins.

Macrophages are the key cells involved in host pathogenicity. Macrophages display phenotypic and functional diversity that can be analysed and detected by proteomic analysis. This article describes how to perform 2D electrophoresis of primary cultures of human macrophages differentiated into M1 or M2 phenotype.

The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The ...

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are able to replicate in the deep lung. Phagocytosis of Cryptococcus by macrophages is one of the ways that the disease is able to spread into the central nervous system to cause lethal meningoencephalitis. Therefore, study of the association between Cryptococcus and macrophages is important to understanding the progression of the infection. The present study describes a step-by-step protocol to study macrophage infectivity by C. neoformansin vitro. Using this protocol, the role of host sterols on host-pathogen interactions is studied. Different concentrations of methyl--cyclodextrin (MCD) were used to deplete cholesterol from murine reticulum sarcoma macrophage-like cell line J774A.1. Cholesterol depletion was confirmed and quantified using both a commercially available cholesterol quantification kit and thin layer chromatography. Cholesterol depleted cells were activated using Lipopolysacharide (LPS) and Interferon gamma (IFN&#947;) and infected with antibody-opsonized Cryptococcus neoformans wild-type H99 cells at an effector-to-target ratio of 1:1. Infected cells were monitored after 2 hr of incubation with C. neoformans and their phagocytic index was calculated. Cholesterol depletion resulted in a significant reduction in the phagocytic index. The presented protocols offer a convenient method to mimic the initiation of the infection process in a laboratory environment and study the role of host lipid composition on infectivity.

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

In this article, a protocol for infection of macrophages with Cryptococcus neoformans is described. Also, a method for sterol depletion from the macrophages is explained. These protocols provide a guide to study fungal infections in vitro and examine the role of sterols in such infections.

Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are ...

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 ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

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

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

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

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

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

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

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

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

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

Tuning Degradation to Achieve Specific and Efficient Protein Depletion

The plant auxin binding receptor, TIR1, recognizes proteins containing a specific auxin-inducible degron (AID) motif in the presence of auxin, targeting them for degradation. This system is exploited in many non-plant eukaryotes, such that a target protein, tagged with the AID motif, is degraded upon auxin addition. The level of TIR1 expression is critical; excessive expression leads to degradation of the AID-tagged protein even in the absence of auxin, whereas low expression leads to slow depletion. A &#946;-estradiol-inducible AID system was created, with expression of TIR1 under the control of a &#946;-estradiol inducible promoter. The level of TIR1 is tunable by changing the time of incubation with &#946;-estradiol before auxin addition. This protocol describes how to rapidly deplete a target protein using the AID system. The appropriate &#946;-estradiol incubation time depends on the abundance of the target protein. Therefore, efficient depletion depends on optimal timing that also minimizes auxin-independent depletion.

Here, we present a protocol to effectively and specifically deplete a protein of interest in the yeast Saccharomyces cerevisiae using the &#946;-est AID system.

The plant auxin binding receptor, TIR1, recognizes proteins containing a specific auxin-inducible degron (AID) motif in the presence of auxin, targeting ...