Name: measuring granulocyte and monocyte phagocytosis and oxidative burst activity in human blood
Uploaded: 2016-09-12T13:00:00.000+00:00
Duration: 11 min 29 s
Description: Watch this Scientific Journal Video about Measuring Granulocyte and Monocyte Phagocytosis and Oxidative Burst Activity in Human Blood at JoVE.com

Os autores gostariam de reconhecer Zack Shue, Casey John e Lynn Cialdella-Kam para a sua assistência durante a otimização deste processo.

<ol>
	<li>Nieman, D. C., 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=Influence+of+pistachios+on+performance+and+exercise-induced+inflammation,+oxidative+stress,+immune+dysfunction,+and+metabolite+shifts+in+cyclists:+a+randomized,+crossover+trial.">Influence of pistachios on performance and exercise-induced inflammation, oxidative stress, immune dysfunction, and metabolite shifts in cyclists: a randomized, crossover trial.</a> PLoS One. 9, e113725 (2014).</li><li>Knab, A. 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=Effects+of+a+freeze-dried+juice+blend+powder+on+exercise-induced+inflammation,+oxidative+stress,+and+immune+function+in+cyclists.">Effects of a freeze-dried juice blend powder on exercise-induced inflammation, oxidative stress, and immune function in cyclists.</a> Appl Physiol Nutr Metab. 39, 381-385 (2014).</li><li>Konrad, 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=The+acute+effect+of+ingesting+a+quercetin-based+supplement+on+exercise-induced+inflammation+and+immune+changes+in+runners.">The acute effect of ingesting a quercetin-based supplement on exercise-induced inflammation and immune changes in runners.</a> Int J Sport Nutr Exerc Metab. 21, 338-346 (2011).</li><li>Nieman, D. C., 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=Immune+and+inflammation+responses+to+a+3-day+period+of+intensified+running+versus+cycling.">Immune and inflammation responses to a 3-day period of intensified running versus cycling.</a> Brain Behav Immun. 39, 180-185 (2014).</li><li>Nieman, D. C., 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=Carbohydrate+supplementation+affects+blood+granulocyte+and+monocyte+trafficking+but+not+function+after+2.5+h+of+running.">Carbohydrate supplementation affects blood granulocyte and monocyte trafficking but not function after 2.5 h of running.</a> Am J Clin Nutr. 66, 153-159 (1997).</li><li>Tidball, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+cell+response+to+acute+muscle+injury.">Inflammatory cell response to acute muscle injury.</a> Med Sci Sports Exerc. 27, 1022-1032 (1995).</li><li>McFarlin, B. K., Williams, R. R., Venable, A. S., Dwyer, K. C., Haviland, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image-based+cytometry+reveals+three+distinct+subsets+of+activated+granulocytes+based+on+phagocytosis+and+oxidative+burst.">Image-based cytometry reveals three distinct subsets of activated granulocytes based on phagocytosis and oxidative burst.</a> Cytometry A. 83, 745-751 (2013).</li><li>Liao, Y., Liu, P., Guo, F., Zhang, Z. Y., Zhang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+burst+of+circulating+neutrophils+following+traumatic+brain+injury+in+human.">Oxidative burst of circulating neutrophils following traumatic brain injury in human.</a> PLoS One. 8, e68963 (2013).</li><li>McFarlin, B. K., Venable, A. S., Prado, E. A., Henning, A. L., Williams, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image-based+flow+cytometry+technique+to+evaluate+changes+in+granulocyte+function+in+vitro.">Image-based flow cytometry technique to evaluate changes in granulocyte function in vitro.</a> J Vis Exp. , (2014).</li><li>Nieman, D. C., 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=Immune+response+to+obesity+and+moderate+weight+loss.">Immune response to obesity and moderate weight loss.</a> Int J Obes Relat Metab Disord. 20, 353-360 (1996).</li><li>Nieman, D. C., 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=Immune+function+in+female+elite+rowers+and+non-athletes.">Immune function in female elite rowers and non-athletes.</a> Br J Sports Med. 34, 181-187 (2000).</li><li>Ichii, H., 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=Iron+sucrose+impairs+phagocytic+function+and+promotes+apoptosis+in+polymorphonuclear+leukocytes.">Iron sucrose impairs phagocytic function and promotes apoptosis in polymorphonuclear leukocytes.</a> Am J Nephrol. 36, 50-57 (2012).</li><li>Gomes, A., Fernandes, E., Lima, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+probes+used+for+detection+of+reactive+oxygen+species.">Fluorescence probes used for detection of reactive oxygen species.</a> J Biochem Biophys Methods. 65, 45-80 (2005).</li><li>Gruger, T., 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=Negative+impact+of+linezolid+on+human+neutrophil+functions+in+vitro.">Negative impact of linezolid on human neutrophil functions in vitro.</a> Chemotherapy. 58, 206-211 (2012).</li><li>Dementhon, K., El-Kirat-Chatel, S., Noel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+an+in+vitro+model+for+the+multi-parametric+quantification+of+the+cellular+interactions+between+Candida+yeasts+and+phagocytes.">Development of an in vitro model for the multi-parametric quantification of the cellular interactions between Candida yeasts and phagocytes.</a> PLoS One. 7, e32621 (2012).</li></ol>

The authors have nothing to disclose.

Este manuscrito fornece um protocolo passo-a-passo para a determinação dos dois indicadores de granulócitos e monócitos. Foram identificadas algumas etapas que são críticos para o sucesso deste ensaio. Um tal passo é a incubação em banho de gelo que ocorre imediatamente antes da adição de bactérias. arrefecimento exaustiva de todas as amostras irá minimizar o efeito da temperatura sobre a actividade de fagocitose e OB. Um outro passo crucial é a adição de bactérias para cada amostra. A 8: 1 bactérias: ...

A explosão de granulócitos e monócitos fagocitose / oxidativo (OB) ensaio da atividade é uma técnica simples e direta que é frequentemente usado para reunir informações sobre a função imunológica inata após o exercício prolongado e intenso. 1-4 Ao estudar a resposta do sistema imunológico a um estímulo, granulócitos e monócitos são de particular interesse porque eles desempenham um papel central na defesa do hospedeiro e são as primeiras células do sistema imunológico a se acumular no local de infecção. 5 neutrófilos, um tipo de granulócitos, são as primeiras células de se translocar para o músculo danificado tecidos após o exercício intenso. 6 fagocitose ea atividade de OB são as medidas mais comuns da função de granulócitos e monócitos, 7 e são preferidos como indicadores da função celular imune inato por causa de seu papel central, tanto no processo de infecção e o processo de reparação. O ensaio descrito no presente Utili manuscritozes um citómetro de fluxo de base para proporcionar uma determinação quantitativa da fagocitose de granulócitos e monócitos e atividade de OB no sangue total. O uso de sangue inteiro nesta técnica é vantajosa porque permite o ensaio a ser conduzido sem um procedimento de purificação que consomem tempo. Este ensaio pode ser facilmente modificado para acomodar uma variedade de modelos de investigação e de capacidades de laboratório. Por exemplo, Liao et al., Utilizou uma técnica semelhante para demonstrar que a actividade dos neutrófilos OB é aumentada e neutrófilos fagocitose é diminuída após lesão cerebral traumática. 8 Num outro exemplo, McFarlin et al. Descrevem uma modificação elegante desta técnica que utiliza aloficocianina (APC ) -conjugated CD66b e de alto desempenho conjunto rotulagem CD45 APC conjugada com a citometria de fluxo baseada em imagem para classificar os granulócitos em termos de suas atividades fagocitose e OB. 7,9 Nosso grupo de pesquisa tem usado várias adaptações desta technique como um indicador da função imune inata em populações com sobrepeso, obesas, e atléticas por quase 20 anos. 10,11 O texto abaixo irá fornecer o leitor com instruções passo-a-passo para a realização deste ensaio e destacar as áreas que o leitor possa se ​​adaptar para atender às necessidades de suas pesquisas.

<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.

o exercício prolongado e intenso tem profundos efeitos sobre a função imunológica inata, incluindo a função natural killer células, resposta mediada por citocinas por macrófagos à infecção viral, e de granulócitos e fagocitose de monócitos e actividade OB. Vários estudos indicam que granulócitos e monócitos fagocitose aumenta significativamente pós-exercício, refletindo a inflamação induzida por lesão muscular e demandas metabólicas. Em contraste, a actividade de gr...

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.

Medição de granulócitos e monócitos fagocitose e oxidativo Atividade Burst no Sangue

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

Research

JoVE Journal

Immunology and Infection

14.1K visualizações.  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.

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

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.

Um<em&gt; In vitro</em&gt; Modelo para estudar a heterogeneidade de diferenciação de macrófagos humanos e Polarização

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

Imunologia e Infecção

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.

Bioenergética e os oxidativo estourou: Protocolos para o Isolamento e Avaliação de leucócitos humanos e plaquetas

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.

Baseada em imagem Técnica de Citometria de Fluxo para avaliar as mudanças na função de granulócitos<em&gt; In Vitro</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 ...

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.

Uso de um monócito monocamada Ensaio para Avaliar a fagocitose mediada pelo receptor Fcy

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.

Time-Lapse imagem em 3D da fagocitose por macrófagos de rato

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

Assessing Respiratory Immune Responses to Haemophilus Influenzae

Haemophilus influenzae (Hi) is a prevalent bacterium found in a range of respiratory conditions. A variety of different assays/techniques may be used to assess the respiratory immune/inflammatory response to this bacterium. Flow cytometry and confocal microscopy are fluorescence-based technologies that allow detailed characterization of biological responses.&#160;Different forms of Hi antigen can be used, including cell wall components, killed/inactivated preparations, and live bacteria. Hi is a fastidious bacterium that requires enriched media but is generally easy to grow in standard laboratory settings. Tissue samples for stimulation with Hi may be obtained from peripheral blood, bronchoscopy, or resected lung (e.g., in patients undergoing surgery for the treatment of lung cancer). Macrophage and neutrophil function may be comprehensively assessed using flow cytometry with a variety of parameters measured, including phagocytosis, reactive oxygen species, and intracellular cytokine production. Lymphocyte function (e.g., T cell and NK cell function) may be specifically assessed using flow cytometry, principally for intracellular cytokine production. Hi infection is a potent inducer of extracellular trap production, both by neutrophils (NETs) and macrophages (METs). Confocal microscopy is arguably the most optimal way to assess NET and MET expression, which may also be used to assess protease activity. Lung immunity to Haemophilus influenzae can be assessed using flow cytometry and confocal microscopy.

Assessing Respiratory Immune Responses to Haemophilus Influenzae

Haemophilus influenzae induces inflammation in the respiratory tract. This article will focus on the use of flow cytometry and confocal microscopy to define immune responses by phagocytes and lymphocytes in response to this bacterium.

Avaliando as respostas imunes respiratórias ao Haemophilus influenzae

Haemophilus influenzae (Hi) is a prevalent bacterium found in a range of respiratory conditions. A variety of different assays/techniques may be used to ...

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).

Previsão da significância do anticorpo eritrocitário usando o ensaio de monócitos-macrófagos

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, ...

Real-Time Imaging of Macrophage Phagocytosis of IgG-Opsonized Red Blood Cells

Source: Horsthemke, M., et al. <a href="https://app.jove.com/v/57566">Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages.</a> J. Vis. Exp. (2018)
This video demonstrates an assay to monitor independent macrophage-mediated red blood cell (RBC) phagocytosis events. Mouse immunoglobulin G (IgG)-opsonized human RBCs are incubated with mouse macrophages and observed under a confocal microscope. The RBCs are engulfed by macrophages via a phagocytic cup formation and targeted for lysosomal degradation.

Imagem em tempo real da fagocitose de macrófagos de glóbulos vermelhos opsonizados por IgG

Source: Horsthemke, M., et al. Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages. J. Vis. Exp. (2018)
This video demonstrates an assay to monitor ...

JoVE Encyclopedia of Experiments

Imunologia

Imunodiagnóstico

Specialized Assays

Quantitative Flow Cytometric Measurement of Fungal Spore Phagocytosis by Human Phagocytes

Source: Hartung, S., et al., <a href="https://app.jove.com/v/60397">Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry.</a> J. Vis. Exp. (2019)
This video demonstrates a method for quantifying the phagocytosis of FITC-labeled pathogenic fungal spores by human leukocytes. Phagocytosed spores are discerned from cell-adherent spores through counterstaining, followed by flow cytometric analysis.

Medição Quantitativa por Citometria de Fluxo de Fagocitose de Esporos Fúngicos por Fagócitos Humanos

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Phagocytosis Assay

	Incubate 2 x ...

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

Source: Meaney, M. P. et al., <a href="https://app.jove.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.

Um ensaio para avaliar a fagocitose e a atividade de explosão oxidativa em granulócitos e monócitos

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

Medição de granulócitos e monócitos fagocitose e oxidativo Atividade Burst no Sangue

Resumo

Explore mais vídeos

Capítulos neste vídeo

Um