Name: functional characterization of regulatory macrophages that inhibit graft-reactive immunity
Uploaded: 2017-06-07T12:30:00
Description: Watch this Scientific Journal Video about Functional Characterization of Regulatory Macrophages That Inhibit Graft-reactive Immunity at JoVE.com

We acknowledge the technical contributions of the Flow Cytometry, Microsurgery, and Bio- repository/Pathology Centers of Research Excellence at Mount Sinai. This work was supported by the COST Action BM1305: Action to Focus and Accelerate Cell Tolerogenic Therapies (A FACTT), the Mount Sinai Recanati/Miller Transplantation Institute developmental funds, Ministerio de Ciencia e Innovacion SAF2013-48834-R and SAF2016-80031-R J.O.

In this study, mice are housed in accordance with the United States Department of Agriculture guidelines and the recommendations of the Public Health Service Guide for the Care and Use of Laboratory Animals. All experimental techniques involving animal use were performed in accordance with Institutional Animal Care and Utilization Committee (IACUC)-approved protocols of the Mount Sinai School of Medicine.
1. Media Preparation
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	<li>Prepare complete RPMI 1640 medium with 10% FBS and 1% pe...

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This protocol describes the methods we used to immunocharacterize graft-infiltrating myeloid cell subsets in an experimental murine model of heart transplantation, which is also applicable to other tissues in different murine experimental models. Low-pressure cell sorting at 20 psi was the preferred method to isolate a good yield of pure cell subsets. Maintaining the purity of each myeloid subset is critical to establish conclusive results of the suppressive capacity between the different myeloid populations. However, ot...

This protocol describes in vitro techniques to study the function of tissue-infiltrating macrophages isolated from cardiac allografts, according to their ability to modulate T-cell responses. Widely described in the literature, fluorescent cell-tracking dyes in combination with flow cytometry, are powerful tools to study the suppressive function of specific cell types in vitro and in vivo. Our protocol follows the carboxyfluorescein succinimidyl ester (CFSE) method for monitoring lymphocyte proliferation in vitro.
When a CFSE-labeled cell divides, its progeny acquires half the number of carboxyfluorescein-tagged molecules6. The corresponding decrease in cell fluorescence by flow cytometry can be used to assess cell division, monitoring the capacity of suppressive macrophages to modulate T-cell immune responses. Since CFSE is a fluorescein-based dye, it is compatible with a broad range of other fluorochromes, making it applicable to multi-color flow cytometry. The appropriate choice of fluorochromes for phenotyping is also important to avoid excessive spectral overlap and the inability to recognize antibody-positive cells, particularly with visible protein dyes such as CFSE7.
There are many advantages of using fluorescent dyes over alternative techniques that measure cell proliferation, such as the thymidine incorporation assay, which uses radiolabeled thymidine (TdR)8. This assay utilizes tritiated thymidine (3H-TdR) that is incorporated into new strands of chromosomal DNA during mitotic cell division. A safety concern associated with this assay is the use of radioisotopes, since a scintillation beta-counter is used to measure the radioactivity in DNA recovered from the cells in order to determine the extent of cell division. Methodologically, the tritiated thymidine assay is not flexible enough to fit important clinical laboratory constraints such as low number of cells and delayed analysis after staining. On the contrary, CFSE staining has been shown to prevent cell proliferation and to interfere with critical activation markers, such as CD69, HLA-DR and CD259. Therefore, understanding advantages and limitations of each methodology, particularly in multicolor studies where multiple dyes are used to track different cell types, is critical for obtaining accurate and reproducible results.

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functional characterization of regulatory macrophages that inhibit graft-reactive immunity

Macrophage accumulation in transplanted organs has long been recognized as a feature of allograft rejection1. Immunogenic monocytes infiltrate the allograft early after transplantation, mount a graft reactive response against the transplanted organ, and initiate organ rejection2. Recent data suggest that suppressive macrophages facilitate successful long-term transplantation3 and are required for the induction of transplantation tolerance4. This suggests a multidimensional concept of macrophage ontogeny, activation, and function, which demands a new roadmap for the isolation and analysis of macrophage function5. Due to the plasticity of macrophages, it is necessary to provide a methodology to isolate and characterize macrophages, depending on the tissue environment, and to define their functions according to different scenarios. Here, we describe a protocol for immune characterization of graft-infiltrating macrophages and the methods we used to functionally evaluate their capacity to inhibit CD8+ T proliferation and to promote CD4+Foxp3+ Treg expansion in vitro.

The representative results show the gating strategy described in the above protocol. Results also display the analysis of the T-cell proliferation activity after the co-culture with graft-infiltrating macrophages. The in vitro suppressive capacity of macrophage subsets was analyzed in Figure 4. The results indicate that the Ly6CloLy6G- macrophages obtained from tolerized recipients are suppressive. The results also indicate that Ly6Cint...

Watch this Scientific Journal Video about Functional Characterization of Regulatory Macrophages That Inhibit Graft-reactive Immunity at JoVE.com

Functional Characterization of Regulatory Macrophages That Inhibit Graft-reactive Immunity

Macrophages are plastic cells of the hematopoietic system that have a crucial role in protective immunity and homeostasis. In this report, we describe optimized in vitro techniques to phenotypically and functionally characterize graft-infiltrating regulatory macrophages that accumulate in the transplanted organ under tolerogenic conditions.

Macrophage accumulation in transplanted organs has long been recognized as a feature of allograft rejection1. Immunogenic monocytes infiltrate the ...

The overall goal of this in vitro procedure is to phenotypically and functionally characterize macrophage populations isolated from allografted hearts in a mass transplantation model. These methods can help answer key questions in the immunity we find about how to improve success in allograph survival and tolerance. The main advantage of this technique is that it allows the study of how a specific tissue infiltrated cardiac allograph isolated microphages subset modulate host tissues response.After generating a single-cell suspension from the allograft, block any non-specific binding on the myeloid cells with one to two microliters of FC receptor blocking monoclonal antibody for 15 minutes. At the end of the incubation, stain the cells with the appropriate fluorescence conjugated myeloid cell surface antibodies for 45 minutes at four degrees Celsius protected from light. Then wash the cells two times in RPMI medium by centrifugation.And re-suspend the pellet in trypan blue for counting. Next, dilute the cells to a one time 10 to the sixth cells per milliliter concentration in fresh medium. And filter the cells through a 70 micron strainer into five-milliliter round-bottom tube.Just before sorting, stain the cells with DAPI as a viability marker and set the flow cytometer to the appropriate macrophage sorting parameters. Place five-milliliter conical tubes containing one milliliter of RPMI medium for collecting the sorted cells and load the sample tube onto the cytometer. In the flow cytometer software, select a blank experiment with a blank panel, and set up a forward versus side scatter dot plot.Gate on the leukocytes, excluding the cell debris and clumps with the lowest forward and side scatter. From this parent gate, create a new dot plot that displays side scatter versus DAPI and gate on the DAPI negative cells. On this newly gated population, create a dot plot that displays CD11b versus CD45, and gate on the CD11b CD45 double positive macrophages and neutrophils.Then create a final dot plot display Ly6C versus Ly6G to allow gating of the desirable population and sort the cells. At the end of the sort, confirm the purity and viability of the samples. Collect the sorted cells by centrifugation, and re-suspend the pellet for counting in fresh RPMI medium.Dilute the macrophage populations to the appropriate concentration for seeding five times 10 to the fourth macrophages per 100 microliters of complete medium per well, in a 96 well round-bottom plate. Then place the cells in a cell culture incubator for at least 24 hours at 37 degrees Celsius and 5%CO2. To isolate the T, put the knock-in mouse in the supine position.First make a mid-line skin incision, carefully cutting the peritoneum and gently spreading apart the skin. Collect the peripheral lymph nodes and the spleen, and place the tissues in a 100-micrometer filter on top of a 50-milliliter conical tube. Using the plunger of a 1-milliliter syringe, gently press the lymphoid tissues through the filter, and rinse the filter with fresh RPMI medium until the mesh is clean.Collect the single-cell suspension by centrifugation, and re-supsend the pellet in two milliliters of ACK lysis buffer. After five minutes at four degrees Celsius, neutralize the lysis buffer with two milliliters of fresh RPMI medium, and centrifuge the cells. Re-suspend the pellet in 200 microliters of fresh medium, and transfer the cells to a five-milliliter round-bottom tube for straining with the appropriate T-cell surface-staining antibodies.After 45 mintues at four degrees Celsius protected from light, wash the cells two times in fresh RPMI medium and count the cells by trypan blue exclusion. Next, dilute the CD8-stained cells to a one times 10 to the sixth cells per milliliter concentration in CFSE for five minutes in a 20-degree Celsius water bath. At the end of the incubation, neutralize the CFSE with two milliliters of RPMI medium, and collect the cells by centrifugation.Re-suspend the cells at one times 10 to the sixth CD8-stained T-cells per milliliter of RPMI medium concentration, and filter all of the cell samples through a 70-micrometer cell strainer into five-milliliter round-bottom tubes. Then label the cells with DAPI and prepare the flow cytometer for sorting as just demonstrated. For CD4 positive T-cell isolation, set up a forward versus side scatter dot plot for gating on the lymphocytes, excluding the debris as well as the large granular myeloid cells.From this parent gate, create a dot plot that displays side scatter versus DAPI, and gate on the DAPI negative cells. On this newly-gated population, create one dot plot that displays side scatter versus CD4, and one dot plot that displays CD8 versus CFSE. Then sort the gated T-cell populations.At the end of the sort, confirm the purity and viability of the samples, and collect the cells by centrifugation for counting. Dilute the cells to a two times 10 to the sixth T-cells per milliliter of RPMI concentration, and 100 microliters of T-cells in complete RPMI medium to each well of macrophages for a 96-hour co-culture at 37 degrees Celsius and 5%CO2. Finally, add five microliters PBS wash T-cell activator CD3 CD28 magnetic beads to each well to activate and expand the T-cells.Ly6Clo Ly6G-macraphages obtained from tolerized recipients exert a suppressive effect on CD8 positive T-cells, with a modest suppressive activity also observed from Ly6C intermediate Ly6CG positive cells. Only Ly6Clo Ly6G-cells promote the in vitro expansion of CD4 positive Foxp3 positive TReg, however. Together, these data support the conclusion that graft infiltrating CD11B positive Ly6Clo Ly6G negative macrophages possess many of the properties reported to be associated with monocyte-derived suppressor cells, including their ability to inhibit CD8 T-cell proliferation and to promote CD4 positive Foxp3 positive TReg expansion.While attempting this procedure, it's important to maintain the purity in order to establish conclusive data about the suppressive capacity of each macrophage subset. Following this procedure, other methods like using commercial enrichment gates can be used for isolating different leukocyte populations of interest. Macrophage sorting is the longest part of this procedure, but when it has been mastered, this part of the technique can be complete in eight hours if performed properly.After its development, this technique paved the way for researchers in the field of immunology to explore the suppressive capacity of infiltrating macrophages in solid organ transplanting sim model. After watching this video, you should have a good understanding of how to characterize different macrophages population in selected from mouse heart allograph.

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Immunology and Infection

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 ...

Forward Genetics Screens Using Macrophages to Identify Toxoplasma gondii Genes Important for Resistance to IFN-&#947;-Dependent Cell Autonomous Immunity

Toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular protozoan pathogen. The parasite invades and replicates within virtually any warm blooded vertebrate cell type. During parasite invasion of a host cell, the parasite creates a parasitophorous vacuole (PV) that originates from the host cell membrane independent of phagocytosis within which the parasite replicates. While IFN-dependent-innate and cell mediated immunity is important for eventual control of infection, innate immune cells, including neutrophils, monocytes and dendritic cells, can also serve as vehicles for systemic dissemination of the parasite early in infection. An approach is described that utilizes the host innate immune response, in this case macrophages, in a forward genetic screen to identify parasite mutants with a fitness defect in infected macrophages following activation but normal invasion and replication in na&#239;ve macrophages. Thus, the screen isolates parasite mutants that have a specific defect in their ability to resist the effects of macrophage activation. The paper describes two broad phenotypes of mutant parasites following activation of infected macrophages: parasite stasis versus parasite degradation, often in amorphous vacuoles. The parasite mutants are then analyzed to identify the responsible parasite genes specifically important for resistance to induced mediators of cell autonomous immunity. The paper presents a general approach for the forward genetics screen that, in theory, can be modified to target parasite genes important for resistance to specific antimicrobial mediators. It also describes an approach to evaluate the specific macrophage antimicrobial mediators to which the parasite mutant is susceptible. Activation of infected macrophages can also promote parasite differentiation from the tachyzoite to bradyzoite stage that maintains chronic infection. Therefore, methodology is presented to evaluate the importance of the identified parasite gene to establishment of chronic infection.

Forward Genetics Screens Using Macrophages to Identify Toxoplasma gondii Genes Important for Resistance to IFN-&#947;-Dependent Cell Autonomous Immunity

Forward genetics is a powerful approach to identify genes in intracellular pathogens important for resistance to cell autonomous immunity. The current approach uses innate immune cells, specifically macrophages, to identify novel Toxoplasma gondii genes important for immune evasion.

Toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular protozoan pathogen. The parasite invades and replicates within ...

Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident immune cell populations in the oral mucosa and gingiva. We have established a technique for the isolation and study of immune cells from murine gingival tissues, an area of constant microbial exposure and a vulnerable site to a common inflammatory disease, periodontitis. Our protocol allows for a detailed phenotypic characterization of the immune cell populations resident in the gingiva, even at steady state. Our procedure also yields sufficient cells with high viability for use in functional studies, such as the assessment of cytokine secretion ex vivo. This combination of phenotypic and functional characterization of the gingival immune cell network should aid towards investigating the mechanisms involved in oral immunity and periodontal homeostasis, but will also advance our understanding of the mechanisms involved in local immunopathology.

We have established a technique for the isolation, phenotypic characterization and functional analysis of immune cells from murine gingiva.

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident ...

Phenotypic and Functional Analysis of Activated Regulatory T Cells Isolated from Chronic Lymphocytic Choriomeningitis Virus-infected Mice

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance and homeostasis maintenance by regulating the immune response. The primary role of Treg cells is to suppress the proliferation of effector T (Teff) cells and the production of cytokines such as IFN-&#947;, TNF-&#945;, and IL-2. It has been demonstrated that Treg cells&#39; ability to inhibit the function of Teff cells is enhanced during persistent pathogen infection and cancer development. To clarify the function of Treg cells under resting or inflamed conditions, a variety of in vitro suppression assays using mouse or human Treg cells have been devised. The main aim of this study is to develop a method to compare the differences in phenotype and suppressive function between resting and activated Treg cells. To isolate activated Treg cells, mice were infected with lymphocytic choriomeningitis virus (LCMV) clone 13 (CL13), a chronic strain of LCMV. Treg cells isolated from the spleen of LCMV CL13-infected mice exhibited both the activated phenotype and enhanced suppressive activity compared with resting Treg cells isolated from na&#239;ve mice. Here, we describe the basic protocol for ex vivo phenotype analysis to distinguish activated Treg cells from resting Treg cells. Furthermore, we describe a protocol for the measurement of the suppressive activity of fully activated Treg cells.

Here, we describe a protocol to analyze the phenotype of regulatory T (Treg) cells isolated from na&#239;ve and chronic lymphocytic choriomeningitis virus-infected mice. In addition, we provide a process to evaluate the suppressive activity of the Treg cells.

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance ...

The Isolation, Differentiation, and Quantification of Human Antibody-secreting B Cells from Blood: ELISpot as a Functional Readout of Humoral Immunity

The hallmark of humoral immunity is to generate functional ASCs, which synthesize and secrete Abs specific to an antigen (Ag), such as a pathogen, and are used for host defense. For the quantitative determination of the functional status of the humoral immune response of an individual, both serum Abs and circulating ASCs are commonly measured as functional readouts. In humans, peripheral blood is the most convenient and readily accessible sample that can be used for the determination of the humoral immune response elicited by host B cells. Distinct B-cell subsets, including ASCs, can be isolated directly from peripheral blood via selection with lineage-specific Ab-conjugated microbeads or via cell sorting with flow cytometry. Moreover, purified na&#239;ve and memory B cells can be activated and differentiated into ASCs in culture. The functional activities of ASCs to contribute to Ab secretion can be quantified by ELISpot, which is an assay that converges enzyme-linked immunoabsorbance assay (ELISA) and western blotting technologies to enable the enumeration of individual ASCs at the single-cell level. In practice, the ELISpot assay has been increasingly used to evaluate vaccine efficacy because of the ease of handling of a large number of blood samples. The methods of isolating human B cells from peripheral blood, the differentiation of B cells into ASCs in vitro, and the employment of ELISpot for the quantification of total IgM- and IgG-ASCs will be described here.

Human peripheral blood is commonly used for the assessment of the humoral immune response. Here, the methods for isolating human B cells from peripheral blood, differentiating human B cells into antibody (Ab)-secreting B cells (ASCs) in culture, and enumerating the total IgM- and IgG-ASCs via an ELISpot assay are described.

The hallmark of humoral immunity is to generate functional ASCs, which synthesize and secrete Abs specific to an antigen (Ag), such as a pathogen, and are ...

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 ...

Using X-ray Crystallography, Biophysics, and Functional Assays to Determine the Mechanisms Governing T-cell Receptor Recognition of Cancer Antigens

Human CD8+ cytotoxic T lymphocytes (CTLs) are known to play an important role in tumor control. In order to carry out this function, the cell surface-expressed T-cell receptor (TCR) must functionally recognize human leukocyte antigen (HLA)-restricted tumor-derived peptides (pHLA). However, we and others have shown that most TCRs bind sub-optimally to tumor antigens. Uncovering the molecular mechanisms that define this poor recognition could aid in the development of new targeted therapies that circumnavigate these shortcomings. Indeed, present therapies that lack this molecular understanding have not been universally effective. Here, we describe methods that we commonly employ in the laboratory to determine how the nature of the interaction between TCRs and pHLA governs T-cell functionality. These methods include the generation of soluble TCRs and pHLA and the use of these reagents for X-ray crystallography, biophysical analysis, and antigen-specific T-cell staining with pHLA multimers. Using these approaches and guided by structural analysis, it is possible to modify the interaction between TCRs and pHLA and to then test how these modifications impact T-cell antigen recognition. These findings have already helped to clarify the mechanism of T-cell recognition of a number of cancer antigens and could direct the development of altered peptides and modified TCRs for new cancer therapies.

Here, we describe methods that we commonly employ in the laboratory to determine how the nature of the interaction between the T-cell receptor and tumor antigens, presented by human leukocyte antigens, governs T-cell functionality; these methods include protein production, X-ray crystallography, biophysics, and functional T-cell experiments.

Human CD8+ cytotoxic T lymphocytes (CTLs) are known to play an important role in tumor control. In order to carry out this function, the cell ...

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 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 ...

Flow Cytometric Measurement Of ROS Production In Macrophages In Response To Fc&#947;R Cross-linking

The oxidative or respiratory burst is used to describe the rapid consumption of oxygen and generation of reactive oxygen species (ROS) by phagocytes in response to various immune stimuli. ROS generated during immune activation exerts potent antimicrobial activity primarily through the ability of ROS to damage DNA and proteins, causing death of microorganisms. Being able to measure ROS production reproducibly and with ease is necessary in order to assess the contribution of various pathways and molecules to this mechanism of host defense. In this paper, we demonstrate the use of fluorescent probes and flow cytometry to detect ROS production. Although widely used, fluorescent measurement of ROS is notoriously problematic, especially with regards to measurement of ROS induced by specific and not mitogenic stimuli. We present a detailed methodology to detect ROS generated as a result of specific Fc&#947;R stimulation beginning with macrophage generation, priming, staining, Fc&#947;R cross-linking, and ending with flow cytometric analysis.

This study demonstrates the use of flow cytometry to detect reactive oxygen species (ROS) production resulting from activation of the Fc&#947;R. This method can be used to assess changes in the antimicrobial and redox signaling function of phagocytes in response to immune complexes, opsonized microorganisms, or direct Fc&#947;R cross-linking.

The oxidative or respiratory burst is used to describe the rapid consumption of oxygen and generation of reactive oxygen species (ROS) by phagocytes in ...