We would like to thank M. H&ouml;xter for technical assistance in sorting primary murine AECII from biosafety level 2 samples.
This work was supported by grants from the German Research Foundation (DFG) to DB (SFB587, TP B12 and BR2221/1-1) and a stipend from the Hannover Biomedical Research School (DFG GSC 108) to AA. DB is supported by the President&acute;s Initiative and Networking Fund of the Helmholtz Association of German Research Centers (HGF) under contract number W2/W3-029.

Details regarding required reagents and materials are listed in the table at the end of the protocol below. Before starting work, prepare 15 ml tubes (one per mouse) containing 4 ml of dispase and pre-warm them to 37 &deg;C in a water bath. In a heating block, shortly heat small aliquots of 1 % low-melt agarose (in water) to 95 &deg;C until liquefied and subsequently cool to 45 &deg;C until use.
1. Preparation of the Mouse Lung
<ol>
	<li>Sacrifice the mouse by CO2 asphyxiation. Note: Do not perform cervical dislocation as this will injure the trachea so that following steps of the protocol, such as installation of the lung with liquid, cannot be performed successfully. Ensure loss of nociceptive reflexes. </li>
	<li>Spray the mouse with ethanol and make a long cut along the ventral midline of the body. Pull the ventral fur and skin and subsequently also carefully cut and remove the peritoneum.</li>
	<li>Exsanguinate the mouse by cutting the left and right jugular vein (Vena jugularis) as well as the renal artery (Arteria renalis). Remove flowing blood with tissue.</li>
	<li>Carefully puncture and then remove the diaphragm to expose the heart and lung by cutting away the ribs. Take special care not to injure the lung.</li>
	<li>With a 26G cannula and a 10 ml syringe filled with cold phosphate buffered saline (PBS), puncture the right ventricle of the heart and perfuse the lung with PBS until free of blood.</li>
	<li>Cut and remove the salivary glands to expose the trachea. Also carefully cut the muscle surrounding the trachea.</li>
	<li>Insert a 22G indwelling cannula into the trachea, remove the needle and push the plastic catheter towards the lung. Fix the catheter by tying a small piece of yarn around trachea and catheter.</li>
</ol>
2. Enzymatic Digestion of the Lung Tissue
If desired, a bronchoalveolar lavage fluid sample can be prepared by flushing the lungs through the catheter inserted in the trachea with PBS or medium before the next step.
<ol>
	<li>Using a 2 ml syringe, carefully instill a maximal volume of 2 ml of dispase (from the 4 ml aliquot) into the lung through the catheter so that all lobes are fully expanded. Exchange the syringe with a 1 ml syringe containing 0.5 ml of the liquefied agarose and also instill into the lung.</li>
	<li>Leave the syringe on the catheter to prevent back-flow of the agarose and immediately cover the lung with laboratory tissue paper and ice. Let the agarose gel for a couple of minutes.</li>
	<li>Remove the tissue paper, ice, syringe and catheter, cut the trachea and excise the lung, heart and thymus from the chest. Then rinse the lung in a dish with PBS and remove the heart, thymus and remaining trachea.</li>
	<li>Put the lung into the remaining 2 ml of dispase and incubate at room temperature for 45 min.</li>
</ol>
3. Preparation of the Lung Cell Suspension
<ol>
	<li>Remove the lung from the dispase and transfer it into a dish containing 7 ml of anti-CD16/32 antibody (1 &mu;g/ml final concentration) in DMEM medium and 100 &mu;l DNase.</li>
	<li>Using forceps, completely disintegrate the lung tissue by pulling it apart and incubate for 10 min at room temperature while gently rocking on a rocker set to 200 rpm. If desired, the cell suspension can then be stored at 4 &deg;C until the next step.</li>
	<li>Filter the cell suspension through nylon meshes first with 100 &mu;m and subsequently with 70 &mu;m, 48 &mu;m and 30 &mu;m pores. Take care to rinse each mesh as well as the tubes and dishes thoroughly with DMEM to minimize loss of cells and maximize yield. If you are pooling samples, this can be achieved here by subsequently passing cells suspensions from mice of one group through the same filter. Renew the filter in case of clogging.</li>
	<li>Depending on the volume, transfer the filtrate to one or more 50 ml tubes and centrifuge for 15 min at 160 x g and 4 &deg;C. Remove the supernatant.</li>
	<li>Resuspend the cell pellet in 2 ml erythrocyte lysis buffer (0.15 M NH4Cl, 0.01 M KHCO3, 0.1 mM EDTA, pH 7.2) and quickly terminate lysis by addition of 13 ml of DMEM medium. Transfer the solution to a 15 ml tube and centrifuge for 12 min at 160 x g and 4 &deg;C.</li>
</ol>
4. Antibody Staining for Flow Cytometric Cell-sorting
<ol>
	<li>Resuspend the cells in 3 ml primary antibody cocktail, prepared in DMEM medium at dilutions suitable for flow cytometry. (Antibodies were previously titrated for optimal working dilutions by staining 1 x 106 splenocytes in a volume of 100 &mu;l. Use 3 ml of the primary antibody cocktail containing the optimal concentrations for each of the used antibodies for cells pooled from up to five mice. If more mice are pooled to one sample, adjust the volume).</li>
	<li>For staining, incubate the cells for 10 min in the dark at 4 &deg;C.</li>
	<li>Wash the cells by filling the 15 ml tube with DMEM medium and centrifugation for 12 min at 160 x g and 4 &deg;C.</li>
	<li>In case uncoupled or biotinylated antibodies were used in 4.1: Remove the supernatant and resuspend the cells in 2 - 3 ml secondary antibody cocktail. Stain for 10 min at 4 &deg;C in the dark.</li>
	<li>Wash the cells through filling the tube with DMEM and centrifugation for 12 min at 160 x g and 4 &deg;C.</li>
	<li>Resuspend the cells in 1 ml of DMEM and pre-filter through a 50 &mu;m filter into a tube for cell-sorting. Optional: Count the cells to obtain the total cell number in the lung cell suspension.</li>
</ol>
5. Cell-sorting
<ol>
	<li>Mix the cells by vortexing prior to sorting. Use a 100 &mu;m nozzle for cell-sorting of AECII. Sheath fluid pressure as well as laser emission and detection wavelengths depend on the instrument used for flow cytometric cell-sorting as well as the fluorochromes used in the antibody staining procedure.</li>
	<li>Gate on the SSChigh cells that are negative for the fluorochromes used for staining and sort into a tube containing DMEM. Use forward scatter height (FSC-H) vs. area (FSC-A) and sideward scatter height (SSC-H) vs. area (SSC-A) windows to exclude any doublets or cell aggregates (see details of the gating strategy in Figure 1a).</li>
	<li>In order to collect the sorted cells centrifuge for 20 min at 280 x g and 4 &deg;C.</li>
	<li>Resuspend the cells in culture-medium or buffer as required for subsequent analysis.</li>
</ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=No+longer+an+innocent+bystander:+epithelial+toll-like+receptor+signaling+in+the+development+of+mucosal+inflammation.">No longer an innocent bystander: epithelial toll-like receptor signaling in the development of mucosal inflammation.</a> Mol. Med. 14, 645-659 (2008).</li><li>Herold, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alveolar+epithelial+cells+direct+monocyte+transepithelial+migration+upon+influenza+virus+infection:+impact+of+chemokines+and+adhesion+molecules.">Alveolar epithelial cells direct monocyte transepithelial migration upon influenza virus infection: impact of chemokines and adhesion molecules.</a> J. Immunol. 177, 1817-1824 (2006).</li><li>Knight, D. A., Holgate, S. 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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin-1beta+regulates+CXCL8+release+and+influences+disease+outcome+in+response+to+Streptococcus+pneumoniae,+defining+intercellular+cooperation+between+pulmonary+epithelial+cells+and+macrophages.">Interleukin-1beta regulates CXCL8 release and influences disease outcome in response to Streptococcus pneumoniae, defining intercellular cooperation between pulmonary epithelial cells and macrophages.</a> Infect. 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No conflicts of interest declared.

Our protocol for the isolation of murine AECII by flow cytometry offers a rapid way of accessing primary cells from the mouse lung for a whole range of functional and molecular studies. The described procedure yields highly viable and pure populations of AECII that are sufficient in number for direct subsequent analyses, such as RNA isolation (see Figure 2b) and transcriptome studies. For functional applications, it is also possible to culture the isolated cells, allowing e.g. the generation of ...

<table border="1">
 <tbody>
 <tr>
 <td>Name of reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>indwelling cannula Introcan 22G</td>
 <td>Braun</td>
 <td>REF 4252098B</td>
 <td></td>
 </tr>
 <tr>
 <td>Dispase, 100 ml(5000 caseinolytic units)</td>
 <td>BD Biosciences</td>
 <td>354235</td>
 <td>aliquot to 4 ml in 15 ml tubes, store at -20 &deg;C</td>
 </tr>
 <tr>
 <td>Biozym Plaque Agarose</td>
 <td>Biozym</td>
 <td>840101</td>
 <td>1% w/v in H2O</td>
 </tr>
 <tr>
 <td>Deoxyribonuclease I from bovine pancreas, 2000 Kunitz units/vial</td>
 <td>Sigma-Aldrich</td>
 <td>D4263</td>
 <td>freshly dissolve content of 1 vial in 300 &mu;l DMEM</td>
 </tr>
 <tr>
 <td>DMEM</td>
 <td>Gibco</td>
 <td>22320-022</td>
 <td>used as provided by manufacturer (Low Glucose, Pyruvate, HEPES)</td>
 </tr>
 <tr>
 <td>cell strainers (100 &mu;m, 75 &mu;m) </td>
 <td>BD Falcon</td>
 <td>352360, 352350</td>
 <td></td>
 </tr>
 <tr>
 <td>nylon mesh(48 &mu;m, 30 &mu;m) </td>
 <td>B&uuml;ckmann GmbH</td>
 <td>03-48/26-1020, 03-30/18-108</td>
 <td></td>
 </tr>
 <tr>
 <td>CellTrics 50 &mu;m filter</td>
 <td>PARTEC</td>
 <td>04-0042-2317</td>
 <td></td>
 </tr>
 <tr>
 <td>anti-mouse CD16/CD32</td>
 <td>BioLegend</td>
 <td>101302</td>
 <td>clone 93; purified</td>
 </tr>
 <tr>
 <td>anti-mouse F4/80</td>
 <td>BioLegend</td>
 <td>123116</td>
 <td>clone BM8; APC coupled</td>
 </tr>
 <tr>
 <td>anti-mouse CD11b</td>
 <td>BioLegend</td>
 <td>101208</td>
 <td>clone M1/70; PE coupled</td>
 </tr>
 <tr>
 <td>anti-mouse CD11c</td>
 <td>BioLegend</td>
 <td>117310</td>
 <td>clone N418; APC coupled</td>
 </tr>
 <tr>
 <td>anti-mouse CD45</td>
 <td>BioLegend</td>
 <td>103102</td>
 <td>clone 30-F11; purified</td>
 </tr>
 <tr>
 <td>anti-mouse CD19</td>
 <td>eBioscience</td>
 <td>12-0193-83</td>
 <td>eBio 1D3; PE coupled</td>
 </tr>
 <tr>
 <td>polyclonal
 goat anti-rat IgG</td>
 <td>BD 
 Pharmingen</td>
 <td>550767</td>
 <td>polyclonal, PE coupled</td>
 </tr>
 <tr>
 	<td></td>
 <td></td>
 <td></td>
 <td>Antibodies coupled to alternative fluorochromes can be used, depending on the flow cytometer and lasers available.
</td>
 </tr>
 </tbody>
</table>

flow cytometric isolation of primary murine type ii alveolar epithelial cells for functional and molecular studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

When sorting lung cell suspensions isolated from healthy mice, the AECII gate will typically account for about 42 &plusmn; 10 % of all events. This percentage can be noticeably lower when mice with respiratory conditions such as a viral infection are used, as the initial cell suspension will contain a considerably higher proportion of lymphocytes and other immune cells recruited to the airways. For AECII isolated from IAV infected lungs on day 3 following infection we have observed a reduction of the frequency of cells i...

Watch this Scientific Journal Video about Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies at JoVE.com

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

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Immunology and Infection

26.2K Views.  Helmholtz Centre for Infection Research. Descriviamo l'isolamento rapido di primarie murine di tipo II le cellule epiteliali alveolari (AECII) per selezione di citometria di flusso negativo. Questi AECII mostrano alta vitalità e purezza e sono adatte per una vasta gamma di studi funzionali e molecolari riguardanti il ​​loro ruolo in condizioni respiratorie come le malattie autoimmuni o infettive.

Video: Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam cells, immune cells, vascular endothelial and smooth muscle cells, platelets, extracellular matrix, and a lipid-rich core with extensive necrosis and fibrosis of surrounding tissues.1 The innate and adaptive arms of the immune response are involved in the initiation, development and persistence of atherosclerosis.2, 3 There is a significant body of evidence that different subsets of the immune cells, such as macrophages, dendritic cells, T and B lymphocytes, are present within the aortas of healthy and atherosclerosis-prone mice4. Additionally, immune cells are found in the surrounding aortic adventitia which suggests an important role of this tissue in atherogenesis.2
For some time, the quantitative detection of different types of immune cells, their activation status, and the cellular composition within the aortic wall was limited by RT-PCR and immunohistochemical methods for the study of atherosclerosis. Few attempts were made to perform flow cytometry using human aortas, and a number of problems, such as a high autofluorescence, have been reported5,6. Human atherosclerotic plaques were digested with collagenase 1, and free cells were collected and stained for CD14+/CD11c+ to highlight macrophage-derived foam cells. In this study, a "mock" channel was used to avoid false-positive staining.6 Necrotic materials accumulating during the digestion process give rise in a large amount of debris that generates a high autofluorescence in aortic samples. To resolve this problem, a panel of negative and positive controls has been proposed, but only double staining could be applied in these samples. We have developed a new flow cytometry-based method7 to analyze the immune cell composition and characterize the activation, proliferation, differentiation of immune cells in healthy and atherosclerosis-prone aorta. This method allows the investigation of the immune cell composition of the aortic wall and opens possibilities to use a broad spectrum of immunological methods for investigations of immune aspects of this disease.

This paper presents a flow cytometry-based method to investigate the immune composition of aortas. The paper also illustrates an additional technique that allows examining surrounding adventitia and vessel wall separately. This method opens possibilities to perform phenotypical analyses of aortic leukocytes and apply several immunological assays for atherosclerosis studies.

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is initiated by interaction of the pathogen with intestinal epithelial cells. This interaction leads to disruption of intercellular structures such as tight junctions (TJ). TJ ensure sealing of the epithelial layer to separate host tissue from gut lumen. Recent studies provide evidence that disruption of TJ by the parasitic protein EhCPADH112 is a prerequisite for E. histolytica invasion that is accompanied by epithelial barrier dysfunction. Thus, the analysis of molecular mechanisms involved in TJ disassembly during E. histolytica invasion is of paramount importance to improve our understanding of amoebiasis pathogenesis. This article presents an easy model that allows the assessment of initial host-pathogen interactions and the parasite invasion potential. Parameters to be analyzed include transepithelial electrical resistance, interaction of EhCPADH112 with epithelial surface receptors, changes in expression and localization of epithelial junctional markers and localization of parasite molecules within epithelial cells.

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Human infection by Entamoeba histolytica leads to amoebiasis, a major cause of diarrhea in tropical countries. Infection is initiated by pathogen interactions with intestinal epithelial cells, provoking the opening of cell-cell contacts and consequently diarrhea, sometimes followed by liver infection. This article provides a model to assess the early host-pathogen interactions to improve our understanding of amoebiasis pathogenesis.

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.
Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%&#177; 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%&#177; 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. 
This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.

Here we present a protocol that generates large amounts of murine monocytes from heterogeneous bone marrow for translational applications. In comparison to others, this new method helps reduce the number of sacrificed animals and lowers costs by avoiding expensive methods such as high gradient magnetic cell separation (MACS).

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 malignant gliomas soon after intracranial engraftment if the cancer cells are rendered deficient in their expression of the &#946;-galactoside-binding lectin galectin-1 (gal-1). More recent work now shows that a population of Gr-1+/CD11b+ myeloid cells is critical to this effect. To better understand the mechanisms by which NK and myeloid cells cooperate to confer gal-1-deficient tumor rejection we have developed a comprehensive protocol for the isolation and analysis of glioma-infiltrating peripheral blood mononuclear cells (PBMC). The method is demonstrated here by comparing PBMC infiltration into the tumor microenvironment of gal-1-expressing GL26 gliomas with those rendered gal-1-deficient via shRNA knockdown. The protocol begins with a description of&#160;how to culture and prepare GL26 cells for inoculation into the syngeneic C57BL/6J mouse brain. It then explains the steps involved in the isolation and flow cytometric analysis of glioma-infiltrating PBMCs from the early brain tumor microenvironment. The method is adaptable to a number of in vivo experimental designs in which temporal data on immune infiltration into the brain is required. The method is sensitive and highly reproducible, as glioma-infiltrating PBMCs can be isolated from intracranial tumors as soon as 24 hr post-tumor engraftment with similar cell counts observed from time point matched tumors throughout independent experiments. A single experimentalist can perform the method from brain harvesting to flow cytometric analysis of glioma-infiltrating PBMCs in roughly 4-6 hr depending on the number of samples to be analyzed. Alternative glioma models and/or cell-specific detection antibodies may also be used at the experimentalists&#8217; discretion to assess the infiltration of several other immune cell types of interest without the need for alterations to the overall procedure.

Presented here is a straightforward method for the isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells that yields time-dependent quantitative data on the number and activation status of immune cells entering the early brain tumor microenvironment.

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 ...

Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both microbial and dietary. Given an increasing understanding that these luminal antigens help shape the immune response and that education of immune cells within the intestine is critical for a number of systemic diseases, there has been increased interest in characterizing the intestinal immune system. However, many published protocols are arduous and time-consuming. We present here a simplified protocol for the isolation of lymphocytes from the small-intestinal lamina propria, intraepithelial layer, and Peyer&#39;s patches that is rapid, reproducible, and does not require laborious Percoll gradients. Although the protocol focuses on the small intestine, it is also suitable for analysis of the colon. Moreover, we highlight some aspects that may need additional optimization depending on the specific scientific question. This approach results in the isolation of large numbers of viable lymphocytes that can subsequently be used for flow cytometric analysis or alternate means of characterization.

There is growing interest in the quantitative characterization of intestinal lymphocytes owing to increasing recognition that these cells play a critical role in a variety of intestinal and systemic diseases. In this protocol, we describe how to isolate single cell populations from different small-intestinal compartments for subsequent flow cytometric characterization.

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both ...

Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore essential for understanding pathogenicity of different pathogens including HIV. Gamma delta (GD) T cells are the prototype of &#39;unconventional&#39; T cells and represent a relatively small subset of T cells defined by their expression of heterodimeric T-cell receptors (TCRs) composed of gamma and delta chains. This sets them apart from the classical and much better known CD4+ helper T cells and CD8+ cytotoxic T cells that are defined by alpha-beta TCRs. GD T cells often show tissue-specific localization and are enriched in epithelium. GD T cells orchestrate immune responses in inflammation, tumor surveillance, infectious disease, and autoimmunity.
Here, we present a method to reproducibly isolate and analyze human endocervical intraepithelial GD T lymphocytes. We have used endocervical cytobrush samples from women participating in the Women&#39;s Interagency HIV Infection Study (WIHS). Knowledge about GD T cells interactions during conditions in which there is an insult to the vaginal mucosal could be applied to any clinical study in which mucosal vulnerability is addressed, including the development of vaginal microbicides.In addition, knowledge about mucosal GD T cell responses has potential for application of GD T cell-based immune therapy in treating infectious diseases.

We describe here an isolation method to obtain human endocervical intraepithelial lymphocytes for the analysis of intraepithelial gamma delta T cells. This protocol can be extended for the purification of endocervical gamma delta T cells by magnetic beads or by cell sorting.

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore ...

Isolation and Characterization of Neutrophil-derived Microparticles for Functional Studies

Polymorphonuclear neutrophil-derived microparticles (PMN)-MPs) are lipid bilayer, spherical microvesicles with sizes ranging from 50&#8211;1,000 nm in diameter. MPs are a newly evolving, important part of cell-to-cell communication and signaling machinery. Because of their size and the nature of their release, until recently MP existence was overlooked. However, with improved technology and analytical methods their function in health and disease is now emerging. The protocols presented here are aimed at isolating and characterizing PMN-MPs by flow cytometry and immunoblotting. Moreover, several implementation examples are given. These protocols for MP isolation are fast, low-cost, and do not require the use of expensive kits. Furthermore, they allow for the labeling of MPs following isolation, as well as pre-labeling of source cells prior to MP release, using a membrane-specific fluorescent dye for visualization and analysis by flow cytometry. These methods, however, have several limitations including purity of PMNs and MPs and the need for sophisticated analytical instrumentation. A high-end flow cytometer is needed to reliably analyze MPs and minimize false positive reads due to noise or auto-fluorescence. The described protocols can be used to isolate and define MP biogenesis, and characterize their markers and variation in composition under different stimulating conditions. Size heterogeneity can be exploited to investigate whether the content of membrane particles versus exosomes is different, and whether they fulfill different roles in tissue homeostasis. Finally, following isolation and characterization of MPs, their function in cellular responses and various disease models (including, PMN-associated inflammatory disorders, such as Inflammatory Bowel Diseases or Acute Lung Injury) can be explored.

New protocols are described here to isolate and characterize microparticles derived from human and mouse neutrophils. These protocols utilize ultracentrifugation, flow cytometry, and immunoblotting techniques to analyze microparticle content, and they can be used to study the role of microparticles derived from various cell types in cellular function.

Polymorphonuclear neutrophil-derived microparticles (PMN)-MPs) are lipid bilayer, spherical microvesicles with sizes ranging from 50&#8211;1,000 nm in ...

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 ...