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<ol>
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The authors have nothing to disclose.

이 원고는 과립구 및 단핵구 함수의 두 개의 지표를 결정하기위한 단계적인 프로토콜을 제공한다. 우리는이 분석의 성공에 중요한 몇 가지 단계를 확인했다. 그러한 단계는 세균의 첨가 직전에 발생 빙욕 배양한다. 모든 샘플을 철저히 냉각 식세포 및 OB 활성에 대한 온도의 영향을 최소화한다. 다른 중요한 단계는 각각의 샘플에 대한 박테리아의 추가이다. 8 : 1 세균 : 식세포 비율이 최적의 결과?...

과립구 및 단핵구 식세포 / 산화 버스트 (OB) 활성 분석은 종종 장시간 집중 운동 다음 선천성 면역 기능에 대한 정보를 수집하는 데 사용되는 간단한 직선적 기술이다. 1-4 면역계의 반응을 연구 할 때 이들이 숙주 방어에서 중요한 역할을 감염 부위에 축적 한 최초의 면역 세포이기 때문에, 자극, 과립구 및 단핵구는 특히 중요하다. 5 호중구 과립구의 일종 손상된 근육시키다 최초 셀은 집중 운동 다음 티슈. 6 탐식 및 OB 활성 과립구 및 단핵구 기능 (7)의 가장 일반적인 방법이며 때문에 감염 과정 및 복구 프로세스 모두에서의 중추적 인 역할의 선천성 면역 세포 기능의 지표로서 바람직하다. 이 원고 utili에서 설명하는 분석ZES 기본적인 플로우 사이토 전혈 과립구 및 단핵구와 OB 탐식 활성의 정량적 측정을 제공한다. 이 분석은 시간 소모적 정제 과정없이 수행 될 수 있기 때문에 이러한 기술에서 전혈의 사용은 유리하다. 이러한 분석을 용이하게 연구 설계 및 실험실 기능의 다양성을 수용하기 위해 수정 될 수있다. 예를 들어, 리아 등. 그 호중구 OB 활동을 표시하는 유사한 기술을 이용 외상성 뇌 손상 후 증가되고 호중구 식세포 감소된다. (8) 또 다른 예에서, McFarlin는 외. 알로 사용하는이 기술은 우아한 변형 (APC 바와 )는 식세포 작용 및 OB 활동의 관점에서 과립구 분류 이미지 기반 유동 세포 계측법으로 CD66b 고성능 직렬 APC 공역 CD45 라벨링 π 공역. 7,9 우리의 연구 그룹은이 테의 다양한 적응을 사용하고있다거의 20 년 동안 과체중 비만, 체육 인구의 선천성 면역 기능의 지표로 chnique. 10, 11 텍스트 아래이 분석을 수행하기위한 단계별 지침과 독자를 제공하고 독자가 적응할 수있는 영역을 강조합니다 연구의 요구를 충족한다.

<table><tbody><tr><td>12 x 75 mm tubes, with cap</td><td>VWR</td><td>20170-579</td><td>You will need 2 tubes per sample (&quot;H&quot; and &quot;F&quot;) plus 3 tubes per batch (for assay controls; &quot;F&quot;, &quot;H&quot;, and &quot;Blood only&quot;).</td></tr><tr><td>PBS (10x), pH 7.4</td><td>Life Technologies</td><td>70011-044</td><td></td></tr><tr><td>Bottle top 0.2 &amp;micro;m cellulose acetate filter (500 ml capacity)</td><td>Fisher Scientific</td><td>09-741-07&amp;nbsp;</td><td></td></tr><tr><td>Glucose, powder</td><td>Life Technologies</td><td>15023-021</td><td></td></tr><tr><td>Citric acid (anhydrous, cell culture tested, plant cell culture tested)</td><td>Sigma-Aldrich</td><td>C2404-100G</td><td></td></tr><tr><td>Sodium citrate tribasic dihydrate</td><td>Sigma-Aldrich</td><td>S4641-25G</td><td></td></tr><tr><td>Dihydroethidium (Hydroethidine) (HE)</td><td>Life Technologies</td><td>D11347</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO) Anhydrous</td><td>Life Technologies</td><td>D12345</td><td></td></tr><tr><td>Staphylococcus aureus&amp;nbsp;(Wood strain without protein A) BioParticles, fluorescein conjugate (FITC)</td><td>Life Technologies</td><td>S2851</td><td></td></tr><tr><td>Staphylococcus aureus&amp;nbsp;(Wood strain without protein A) BioParticles, unlabeled</td><td>Life Technologies</td><td>S-2859</td><td></td></tr><tr><td>Trypan Blue Solution, 0.4%</td><td>Life Technologies</td><td>15250-061</td><td></td></tr><tr><td>4.0 ml vacutainer containing 7.2 mg K&lt;sub&gt;2&lt;/sub&gt;EDTA, spray-dried</td><td>VWR</td><td>BD367861</td><td>You will need 1 K&lt;sub&gt;2&lt;/sub&gt;EDTA blood collection tube per sample.</td></tr><tr><td>4.0 ml vacutainer containing 68 USP units Lithium Heparin, spray-coated</td><td>VWR</td><td>BD367884</td><td>You will need 1 Lithium Heparin blood collection tube per sample.</td></tr><tr><td>COULTER Ac&amp;middot;T 5diff CP</td><td>Beckman Coulter</td><td>6605705</td><td>&lt;em&gt;i.e.&lt;/em&gt;, hematology analyzer</td></tr><tr><td>COULTER Ac&amp;middot;T 5diff Rinse</td><td>Beckman Coulter</td><td>8547167</td><td></td></tr><tr><td>COULTER Ac&amp;middot;T 5diff Fix</td><td>Beckman Coulter</td><td>8547171</td><td></td></tr><tr><td>COULTER Ac&amp;middot;T 5diff WBC Lyse</td><td>Beckman Coulter</td><td>8547170</td><td></td></tr><tr><td>COULTER Ac&amp;middot;T 5diff Hgb Lyse</td><td>Beckman Coulter</td><td>8547168</td><td></td></tr><tr><td>COULTER Ac&amp;middot;T 5diff Cal Calibrator</td><td>Beckman Coulter</td><td>7547175</td><td></td></tr><tr><td>COULTER Ac&amp;middot;T 5diff Control Plus</td><td>Beckman Coulter</td><td>7547198</td><td></td></tr><tr><td>AcT 5diff Diluent</td><td>Beckman Coulter</td><td>8547169</td><td></td></tr><tr><td>200 &amp;micro;l extended-length pipette tips</td><td>VWR</td><td>37001-526</td><td></td></tr><tr><td>Insulated ice pan</td><td>VWR</td><td>89198-980</td><td>For ice water bath.</td></tr><tr><td>Open metal tube rack</td><td>VWR</td><td>60916-702</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Life Technologies</td><td>26140-111</td><td></td></tr><tr><td>TQ-Prep Workstation</td><td>Beckman Coulter</td><td>6605429</td><td>&lt;em&gt;i.e.&lt;/em&gt;, automated cell lyse preparation workstation</td></tr><tr><td>ImmunoPrep Reagent System</td><td>Beckman Coulter</td><td>7546999</td><td>&lt;em&gt;i.e.&lt;/em&gt;, RBC lyse/WBC fix reagent system</td></tr><tr><td>Cytomics FC500 MCL Flow Cytometry System with CXP Software</td><td>Beckman Coulter</td><td>626553</td><td></td></tr><tr><td>IsoFlow Sheath Fluid</td><td>Beckman Coulter</td><td>8547008</td><td></td></tr><tr><td>COULTER CLENZ</td><td>Beckman Coulter</td><td>8546929</td><td></td></tr><tr><td>Flow-Check Fluorospheres&amp;nbsp;</td><td>Beckman Coulter</td><td>6605359</td><td>&lt;em&gt;i.e.&lt;/em&gt;, alignment and fluidics verification fluorospheres</td></tr><tr><td>FlowJo Software</td><td>FlowJo</td><td>FlowJo</td><td>&lt;em&gt;i.e.&lt;/em&gt;, flow cytometry analysis software</td></tr><tr><td>Excel Software</td><td>Microsoft</td><td>Microsoft Excel</td><td>&lt;em&gt;i.e.&lt;/em&gt;, electronic spreadsheet program</td></tr></tbody></table>

measuring granulocyte and monocyte phagocytosis and oxidative burst activity in human blood

The granulocyte and monocyte phagocytosis and oxidative burst (OB) activity assay can be used to study the innate immune system. This manuscript provides the necessary methodology to add this assay to an exercise immunology arsenal. The first step in this assay is to prepare two aliquots ("H" and "F") of whole blood (heparin). Then, dihydroethidium is added to the H aliquot, and both aliquots are incubated in a warm water bath followed by a cold water bath. Next, Staphylococcus aureus (S. aureus) is added to the H aliquot and fluorescein isothiocyanate-labeled S. aureus is added to the F aliquot (bacteria:phagocyte = 8:1), and both aliquots are incubated in a warm water bath followed by a cold water bath. Then, trypan blue is added to each aliquot to quench extracellular fluorescence, and the cells are washed with phosphate-buffered saline. Next, the red blood cells are lysed, and the white blood cells are fixed. Finally, a flow cytometer and appropriate analysis software are used to measure granulocyte and monocyte phagocytosis and OB activity. This assay has been used for over 20 years. After heavy and prolonged exertion, athletes experience a significant but transient increase in phagocytosis and an extended decrease in OB activity. The post-exercise increase in phagocytosis is correlated with inflammation. In contrast to normal weight individuals, granulocyte and monocyte phagocytosis is chronically elevated in overweight and obese participants, and is modestly correlated with C-reactive protein. In summary, this flow cytometry-based assay measures the phagocytosis and OB activity of phagocytes and can be used as an additional measure of exercise- and obesity-induced inflammation.

장기간 집중적 인 운동은 자연 살해 세포 기능, 바이러스 성 감염에 대 식세포 사이토 카인 매개 응답 및 과립구와 단핵구의 식균 작용 및 OB 활동을 포함한 선천성 면역 기능에 심각한 영향을 보유하고 있습니다. 여러 연구는 근육 손상 및 신진 대사 요구에 의해 유도 된 염증을 반영하는 과립구와 단핵 탐식의 대폭 증가 후 운동을 나타냅니다. 반면, 과립구와 단핵구 OB 활...

Watch this Scientific Journal Video about Measuring Granulocyte and Monocyte Phagocytosis and Oxidative Burst Activity in Human Blood at JoVE.com

Measuring Granulocyte and Monocyte Phagocytosis and Oxidative Burst Activity in Human Blood

The purpose of this manuscript is to present a method for measuring monocyte and granulocyte phagocytosis and oxidative burst activity in human blood samples.

인간의 혈액에서 측정 과립구와 단핵구 식균 작용 및 산화 버스트 활동

The granulocyte and monocyte phagocytosis and oxidative burst (OB) activity assay can be used to study the innate immune system. This manuscript provides ...

measuring-granulocyte-monocyte-phagocytosis-oxidative-burst-activity

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

14.1K 조회수.  Appalachian State University. The purpose of this manuscript is to present a method for measuring monocyte and granulocyte phagocytosis and oxidative burst activity in human blood samples.

비디오: Measuring Granulocyte and Monocyte Phagocytosis and Oxidative Burst Activity in Human Blood

Proteomic Analysis of Human Macrophage Polarization Under a Low Oxygen Environment

Macrophages are innate immune cells involved in a number of physiological functions ranging from responses to infectious pathogens to tissue homeostasis. The various functions of these cells are related to their activation states, which is also called polarization. The precise molecular description of these various polarizations is a priority in the field of macrophage biology. It is currently acknowledged that a multidimensional approach is necessary to describe how polarization is controlled by environmental signals. In this report, we describe a protocol designed to obtain the proteomic signature of various polarizations in human macrophages. This protocol is based on a label-free quantification of macrophage protein expression obtained from in-gel fractionated and Lys C/trypsin-digested cellular lysis content. We also provide a protocol based on in-solution digestion and isoelectric focusing fractionation to use as an alternative. Because oxygen concentration is a relevant environmental parameter in tissues, we use this protocol to explore how atmospheric composition or a low oxygen environment affects the classification of macrophage polarization.

We present a protocol to obtain proteomic signatures of human macrophages and apply this to determination of the impact of a low oxygen environment on macrophage polarization.

낮은 산소 환경에서 인간 대 식 세포 분극의 Proteomic 분석

Macrophages are innate immune cells involved in a number of physiological functions ranging from responses to infectious pathogens to tissue homeostasis. ...

연구

생화학

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the phenotypic and functional differences between murine and human monocyte-derived macrophages. Therefore, we here describe an in vitro model to generate and study primary human macrophages. Briefly, after density gradient centrifugation of peripheral blood drawn from a forearm vein, monocytes are isolated from peripheral blood mononuclear cells using negative magnetic bead isolation. These monocytes are then cultured for six days under specific conditions to induce different types of macrophage differentiation or polarization. The model is easy to use and circumvents the problems caused by species-specific differences between mouse and man. Furthermore, it is closer to the in vivo conditions than the use of immortalized cell lines. In conclusion, the model described here is suitable to study macrophage biology, identify disease mechanisms and novel therapeutic targets. Even though not fully replacing experiments with animals or human tissues obtained post mortem, the model described here allows identification and validation of disease mechanisms and therapeutic targets that may be highly relevant to various human diseases.

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages are important cells of the innate immune system. Here, we describe an easy to use in vitro model to generate these cells. Using gradient centrifugation, negative bead isolation and specific cell culture conditions, monocyte-derived macrophages can be generated for phenotypic and functional studies.

<em&gt; 체외</em인간 대식 세포의 분화와 양극화의 이질성을 연구&gt; 모델

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the ...

면역학 및 감염병학

Bioenergetics and the Oxidative Burst: Protocols for the Isolation and Evaluation of Human Leukocytes and Platelets

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and neurodegenerative diseases but assessing changes in bioenergetic function in patients is challenging.&#160;Although diseases such as diabetes or atherosclerosis present clinically with specific organ impairment, the systemic components of the pathology, such as hyperglycemia or inflammation, can alter bioenergetic function in circulating leukocytes or platelets. This concept has been recognized for some time but its widespread application has been constrained by the large number of primary cells needed for bioenergetic analysis.&#160;This technical limitation has been overcome by combining the specificity of the magnetic bead isolation techniques, cell adhesion techniques, which allow cells to be attached without activation to microplates, and the sensitivity of new technologies designed for high throughput microplate respirometry.&#160;An example of this equipment is the extracellular flux analyzer. Such instrumentation typically uses oxygen and pH sensitive probes to measure rates of change in these parameters in adherent cells, which can then be related to metabolism. Here we detail the methods for the isolation and plating of monocytes, lymphocytes, neutrophils and platelets, without activation, from human blood and the analysis of mitochondrial bioenergetic function in these cells. In addition, we demonstrate how the oxidative burst in monocytes and neutrophils can also be measured in the same samples. Since these methods use only 8-20 ml human blood they have potential for monitoring reactive oxygen species generation and bioenergetics in a clinical setting.

Blood leukocytes and platelets can be used as a marker of overall bioenergetic health of an individual and so have the potential to monitor pathological processes and the impact of treatments. Here&#160;we describe a method to isolate and measure mitochondrial function and the oxidative burst in these cells.

인간 백혈구 및 혈소판의 분리 및 평가를위한 프로토콜 : 생물 에너지와 산화 버스트

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and ...

Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro

Granulocytes play a key role in the body&#8217;s innate immune response to bacterial and viral infections. While methods exist to measure granulocyte function, in general these are limited in terms of the information they can provide. For example, most existing assays merely provide a percentage of how many granulocytes are activated following a single, fixed length incubation. Complicating matters, most assays focus on only one aspect of function due to limitations in detection technology. This report demonstrates a technique for simultaneous measurement of granulocyte phagocytosis of bacteria and oxidative burst. By measuring both of these functions at the same time, three unique phenotypes of activated granulocytes were identified: 1) Low Activation (minimal phagocytosis, no oxidative burst), 2) Moderate Activation (moderate phagocytosis, some oxidative burst, but no co-localization of the two functional events), and 3) High Activation (high phagocytosis, high oxidative burst, co-localization of phagocytosis and oxidative burst). A fourth population that consisted of inactivated granulocytes was also identified. Using assay incubations of 10, 20, and 40-min the effect of assay incubation duration on the redistribution of activated granulocyte phenotypes was assessed. A fourth incubation was completed on ice as a control. By using serial time incubations, the assay may be able to able to detect how a treatment spatially affects granulocyte function. All samples were measured using an image-based flow cytometer equipped with a quantitative imaging (QI) option, autosampler, and multiple lasers (488, 642, and 785 nm).

Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro

This method demonstrates a technique for assessing granulocyte function by simultaneously measuring phagocytosis of bacteria and oxidative burst. Image-based flow cytometry allowed for the identification of three distinct subsets of activated granulocytes that all differed in their relative functional capacity.

이미지 기반 유동 세포 계측법 기술은 과립구 기능의 변화를 평가하기<em&gt; 체외</em

Granulocytes play a key role in the body&#8217;s innate immune response to bacterial and viral infections. While methods exist to measure granulocyte ...

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

Neutrophils (PMN) are best known for their phagocytic functions against invading pathogens and microorganisms. They have the shortest half-life amongst leukocytes and in their non-activated state are constitutively committed to apoptosis. When recruited to inflammatory sites to resolve inflammation, they produce an array of cytotoxic molecules with potent antimicrobial killing. Yet, when these powerful cytotoxic molecules are released in an uncontrolled manner they can damage surrounding tissues. In recent years however, neutrophil versatility is increasingly evidenced, by demonstrating plasticity and immunoregulatory functions. We have recently identified a new neutrophil-derived subpopulation, which develops spontaneously in standard culture conditions without the addition of cytokines/growth factors such as granulocyte colony-stimulating factor (GM-CSF)/interleukin (IL)-4. Their phagocytic abilities of neutrophil remnants largely contribute to increase their size immensely; therefore they were termed giant phagocytes (G&#981;). Unlike neutrophils, G&#981; are long lived in culture. They express the cluster of differentiation (CD) neutrophil markers CD66b/CD63/CD15/CD11b/myeloperoxidase (MPO)/neutrophil elastase (NE), and are devoid of the monocytic lineage markers CD14/CD16/CD163 and the dendritic CD1c/CD141 markers. They also take-up latex and zymosan, and respond by oxidative burst to stimulation with opsonized-zymosan and PMA. G&#981; also express the scavenger receptors CD68/CD36, and unlike neutrophils, internalize oxidized-low density lipoprotein (oxLDL). Moreover, unlike fresh neutrophils, or cultured monocytes, they respond to oxLDL uptake by increased reactive oxygen species (ROS) production. Additionally, these phagocytes contain microtubule-associated protein-1 light chain 3B (LC3B) coated vacuoles, indicating the activation of autophagy. Using specific inhibitors it is evident that both phagocytosis and autophagy are prerequisites for their development and likely NADPH oxidase dependent ROS. We describe here a method for the preparation of this new subpopulation of long-lived, neutrophil-derived phagocytic cells in culture, their identification and their currently known characteristics. This protocol is essential for obtaining and characterizing G&#981; in order to further investigate their significance and functions.

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

We describe here a method for obtaining and identifying a newly characterized subpopulation of neutrophil-derived giant phagocytes. These cells develop in culture from fresh human blood neutrophils, and are characterized by phagocytosis, autophagy, immensely large size, and extended lifespan. This method is essential to further investigate this unique neutrophil-derived subpopulation.

인간 호중구 유래 거 식세포의 소설 모집단의 개발 및 식별<em&gt; 체외</em

Neutrophils (PMN) are best known for their phagocytic functions against invading pathogens and microorganisms. They have the shortest half-life amongst ...

Use of a Monocyte Monolayer Assay to Evaluate Fc&#947; Receptor-mediated Phagocytosis

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly versatile in vitro assay that can be modified to examine different aspects of antibody and Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis in both research and clinical settings. The assay utilizes adherent monocytes from peripheral blood mononuclear cells isolated from mammalian whole blood. MMA has been described for use in both human and murine investigations. These monocytes express Fc&#947;Rs (e.g., Fc&#947;RI, Fc&#947;RIIA, Fc&#947;RIIB, and Fc&#947;RIIIA) that are involved in immune responses. The MMA exploits the mechanism of Fc&#947;R-mediated interactions, phagocytosis in particular, where antibody-sensitized red blood cells (RBCs) adhere to and/or activate Fc&#947;Rs and are subsequently phagocytosed by the monocytes. In vivo, primarily tissue macrophages found in the spleen and liver carry out Fc&#947;R-mediated phagocytosis of antibody-opsonized RBCs, causing extravascular hemolysis. By evaluating the level of phagocytosis using the MMA, different aspects of the in vivo Fc&#947;R-mediated process can be investigated. Some applications of the MMA include predicting the clinical relevance of allo- or autoantibodies in a transfusion setting, assessing candidate drugs that promote or inhibit phagocytosis, and combining the assay with fluorescent microscopy or traditional Western immunoblotting to investigate the downstream signaling effects of Fc&#947;R-engaging drugs or antibodies. Some limitations include the laboriousness of this technique, which takes a full day from start to finish, and the requirement of research ethics approval in order to work with mammalian blood. However, with diligence and adequate training, the MMA results can be obtained within a 24-h turnover time.

The monocyte monolayer assay (MMA) is an in vitro assay that utilizes isolated primary monocytes obtained from mammalian peripheral whole blood to evaluate Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis.

단핵구 단층 분석의 사용은 Fcγ 수용체 - 매개 식균 작용을 평가하기

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly ...

Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of the actin cytoskeleton to capture, envelop and engulf large particles. Although phagocytic receptors, downstream signaling pathways, and effectors, such as Rho GTPases, have been identified, the dynamic cytoskeletal remodeling of specific receptor-mediated phagocytic events remain unclear. Four decades ago, two distinct mechanisms of phagocytosis, exemplified by Fc&#947; receptor (Fc&#947;R)- and complement receptor (CR)-mediated phagocytosis, were identified using scanning electron microscopy. Binding of immunoglobulin G (IgG)-opsonized particles to Fc&#947;Rs triggers the protrusion of thin membrane extensions, which initially form a so-called phagocytic cup around the particle before it becomes completely enclosed and retracted into the cell. In contrast, complement opsonized particles appear to sink into the phagocyte following binding to complement receptors. These two modes of phagocytosis, phagocytic cup formation and sinking in, have become well established in the literature. However, the distinctions between the two modes have become blurred by reports that complement receptor-mediated phagocytosis may induce various membrane protrusions. With the availability of high resolution imaging techniques, phagocytosis assays are required that allow real-time 3D (three dimensional) visualization of how specific phagocytic receptors mediate the uptake of individual particles. More commonly used approaches for the study of phagocytosis, such as end-point assays, miss the opportunity to understand what is happening at the interface of particles and phagocytes. Here we describe phagocytic assays, using time-lapse spinning disk confocal microscopy, that allow 3D imaging of single phagocytic events. In addition, we describe assays to unambiguously image Fc&#947; receptor- or complement receptor-mediated phagocytosis.

Here we describe methods using spinning disk confocal microscopy to image single phagocytic events by mouse resident peritoneal macrophages. The protocols can be extended to other phagocytic cells.

마우스 대 식 세포에 의해 식 균 작용의 시간 경과 3D 영상

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of ...

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.

선생님 FcγR Cross-linking에 대 식 세포에서 생산의 흐름 Cytometric 측정

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

Prediction of Red Blood Cell Antibody Significance Using the Monocyte-Macrophage Assay

Derived from monocytes in the bone marrow, macrophages are large, innate immune cells that play a major role in clearing dead cells, debris, tumor cells, and foreign pathogens. The phagocytic capacity of monocytes versus macrophages is a concept that is not well understood. Here, we aim to examine a difference in the phagocytosis of monocytes versus macrophages, specifically M1/M2 macrophages, against various opsonized red cells using a modified and updated version of the established monocyte monolayer assay (MMA). Peripheral blood mononuclear cells (PBMCs) were isolated from donor buffy coats. Using purified monocytes, inflammatory M1 and anti-inflammatory M2 macrophages were produced by in vitro culture and polarization. M1/M2 cells were harvested and used in an MMA-like assay, which we refer to as the M-MA, to decipher clinically significant phagocytosis of various red cell antibodies. A phagocytic index (PI) &#62; 5 was deemed clinically significant phagocytosis with the use of monocytes. A phagocytic index (PI) &#62; 12 was deemed clinically significant phagocytosis with the use of M1/M2 macrophages. M2 macrophages demonstrate an increased ability to phagocytose opsonized RBCs compared to monocytes and M1s. The same weak antibody (anti-S) yields significant phagocytosis with only M2 macrophages (PI=43) but not M1s (PI=2) or monocytes (PI=0), and this was demonstrated repeatedly using various antibodies. The use of M2 macrophages instead of monocytes may allow for more accurate results as these cells are more phagocytic, offering further clinical relevance to the assay. Further studies with different antibodies to red blood cells, including validation of the monocyte-macrophage assay (M-MA) with antibodies having known clinical significance, may show the M-MA&#160;more useful to help predict clinically significant red cell alloantibodies and transfusion reactions. This method will advance the field of transfusion medicine and immunology.

We have developed enhancements and updated methods for the existing monocyte-monolayer assay (MMA), in which macrophages are used to help better predict the clinical relevance of red cell alloantibodies in transfusion medicine and immunology. This assay is named the monocyte-macrophage assay (M-MA).

Monocyte-Macrophage Assay를 이용한 적혈구 항체 유의성 예측(Prediction of Red Blood Cell Antibody Significance Using the Monocyte-Macrophage Assay)

Derived from monocytes in the bone marrow, macrophages are large, innate immune cells that play a major role in clearing dead cells, debris, tumor cells, ...

An Assay to Assess Phagocytosis and Oxidative Burst Activity In Granulocytes and Monocytes

Source: Meaney, M. P. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/54264">Measuring Granulocyte and Monocyte Phagocytosis and Oxidative Burst Activity in Human Blood.</a> J. Vis. Exp. (2016)
This video demonstrates the assay to confirm phagocytic activity by cells in human blood via flow cytometry. The granulocytes and monocytes confirm their phagocytic activity by showing green and red fluorescence confirming the presence of engulfed bacteria and oxidative burst activity respectively.

An Assay to Assess Phagocytosis and Oxidative Burst Activity In Granulocytes and Monocytes(과립구 및 단핵구에서 식세포작용 및 산화 버스트 활성을 평가하기 위한 분석)

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human ...