Name: isolation of murine peritoneal macrophages to carry out gene expression analysis upon toll-like receptors stimulation
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Este trabalho foi apoiado por uma bolsa da Ciências Naturais e do Conselho de Investigação do Canadá Engenharia (NSERC, conceder nenhuma 298.515-2011). AL é o destinatário de um Ph.D. bolsa de estudos das ciências naturais e Council of Canada (NSERC) Pesquisa de Engenharia, e MS foi apoiada de uma concessão dos Institutos Canadenses de Pesquisa em Saúde (CIHR, conceder nenhuma. MOP123246).

Todos os procedimentos foram realizados de acordo com a Canadian Council on Animal Care orientações após a aprovação pelo Comitê Animal Care Institucional do Centro de recherche du Centre Hospitalier de l&#39;Université de Montréal (CRCHUM). 1. Isolamento, identificação e Cultura de Murino macrófagos Peritoneal <ol><li> Prepare meio de tioglicolato de 3,8% de cerveja. Para fazê-lo, suspensão 38 g de tioglicolato em 1000 ml de água destilada. Traga solução para ferver para dissolver o meio completamente. Esterilizar por autoclavagem a 121 ° C durante 15 min. Armazenar até 3 meses no escuro, à temperatura ambiente 18. NOTA: Descarte se desenvolve turbidez o que indica uma contaminação bacteriana. A solução pode ser mantida por até um ano no escuro, se manteve estéril. </li><li> Usando seringas de 1 ml ligados a 23 agulhas G, injectar 1 ml de 3,8% de tioglicolato Brewer para dentro da cavidade peritoneal de cada mouse e esperar por 3 dias. Use nova seringa e agulha para cada rato. </li><li> Anestesiar ratos por injecção intraperitoneal de pentobarbital de sódio (100 mg / kg) e a eutanásia ratos por deslocamento cervical. Confirme anestesia adequada, verificando a freqüência respiratória. Normalmente respirações rápidas indicam que o mouse não está profundamente anestesiados. </li><li> Lavar o abdómen de cada rato com etanol a 70%. Usando uma tesoura, realizar uma incisão lateral ao longo da linha média inferior do peritônio. </li><li> Usando uma pinça, puxar para trás a pele abdominal para expor a pele peritoneal transparente. Usando 5 ml seringas unidas a 20 agulhas G, injectar 5 ml de DPBS frio para dentro da cavidade peritoneal de cada rato. </li><li> Realizar uma massagem suave na cavidade peritoneal e, em seguida, aspirar o fluido com cuidado, sem perfurar qualquer órgão. Remover a agulha e dispensar o fluido peritoneal em tubos de 50 ml de centrífuga cónicos. </li><li> Centrifugar durante 10 minutos a 400 xg numa centrifugadora refrigerada.As células devem ficar frio durante todo o procedimento. Descartar o sobrenadante e ressuspender o sedimento de células em meio RPMI 1640. </li><li> Usando um hemocitómetro, contagem de células e ajustar a densidade celular para 1 x 10 6 células / ml. </li><li> Caracterizar o fenótipo de células isoladas por citometria de fluxo utilizando 1 x 10 6 culas por rato e antibodie contra F4 / 80 (um antigénio de superfície expresso em macrófagos). </li></ol> 2. tratamentos com células- <ol><li> Directamente, após isolamento, adicionar 1 x 10 6 células em cada poço. Deixar os macrófagos peritoneais murinos para aderir em placas de 6 poços por cultura durante 1 a 2 horas a 37 ° C. Remover as células não aderentes por lavagem suave três vezes com PBS quente. </li><li> Subsequentemente, as células de cultura em 900 ul de DMEM isento de soro durante 24 horas na presença das seguintes ligandos de TLR: (Pam3CSK4-TLR1 / 2 (0,5 mg / ml); poli (I: C) -TLR3 (10 mg / ml) ; LPS-TLR4 (100 ng / mL); A flagelina-TLR5 (100 ng / ml); FSL1-TLR6 / 2 (100 ng / ml); ssRNA40-TLR7 (1 ug / ml); ODN1826-TLR9 (1 uM). NOTA: Prepare-se para cada ligando uma solução 10x estoque. Adicionar 100 ul desta última em cada poço, realizando assim uma diluição de 10 vezes. </li></ol> 3. Isolamento de ARN <ol><li> Remover todas as células de média e lisam directamente nas placas de seis poços por adição de 1 ml de TRIzol a cada poço e a passagem do ligado de células várias vezes através de uma pipeta. Incubar as amostras homogeneizadas durante 5 a 10 min à temperatura ambiente, para permitir a dissociação completa do complexo de nucleoproteínas. </li><li> Transferir ligado em 1,5 ml RNase- e microtubos livre de DNase. Adicionar 0,2 ml de clorofórmio por cada 1 mL de TRIzol. Agitar tubos vigorosamente à mão durante 15 segundos e incubar à temperatura ambiente durante 5 a 10 min. Centrifuga-se a 12000 xg durante 15 min a 4 ° C. Observe-se a mistura para uma fase de separação vermelho (fenol-clorofórmio) inferior, uma interfase e uma fase aquosa superior incolor. ARN permanece exclusivamente na fase aquosa. </li><li> Transfer cuidadosamente a fase aquosa superior para um tubo fresco, sem perturbar a interfase. Precipitar o ARN a partir dele por mistura com 0,5 ml de álcool isopropílico. Incubar as amostras à temperatura ambiente durante 10 min e centrifuga-se a 12.000 xg durante 10 min a 4 ° C. O ARN precipitados formando um sedimento branco no lado e no fundo do tubo. </li><li> Remover o sobrenadante. Lava-se a pelete de ARN, uma vez com 1 ml de etanol a 75%. Misturar as amostras com vórtex e centrifugar a 7500 xg durante 5 min a 4 ° C. Resumidamente secar o ARN (secar ao ar durante 10 min). Dissolve-se o ARN em 0,1 mL de DEPC (ARNase isenta de água) e incubar durante 10 min a 55 ° C. </li><li> Quantificar ARN utilizando um espectrofotómetro. Para fazê-lo, amostras diluídas 100 vezes em água com DEPC e lidas a comprimentos de onda de 260 nm. A concentração de RNA será então: OD 260 x 40 ng / ul x fator de diluição. </li><li> Equalizar as concentrações de RNA e síntese de cDNA, conforme as instruções do fabricante e utilizando RT-PCR Sistema de kit de primeiros-Strand cDNA Synthesis. Comoacordo com as instruções do fabricante, medir os níveis de ARNm do gene por PCR em tempo real (45 ciclos) em um sistema de detecção de ADN em tempo real. Normalizar os níveis de expressão gênica com gene dois arrumação por exemplo β-actina e GAPDH. NOTA: A concentração de RNA Normalizar a 500 ug / ml e 0,5 ug utilizar para a síntese de cDNA. </li></ol>

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The authors have no competing financial interests.

Os macrófagos são cruciais para a sobrevivência e fornecer um alvo tentador para manipular o host para objectivos imunológicos. A descoberta dos TLRs e outras moléculas de reconhecimento têm conduzido os macrófagos para o centro do debate imunológica. Os macrófagos responder a uma variedade de estímulos, incluindo citocinas, moléculas associadas a danos moleculares padrão (20) amortece e moléculas associadas com grupos de patógenos (PAMPs) 21. Estas respostas estímulos diferentes rep...

O sistema reticuloendotelial fagocítico é composto de células em vários tecidos e órgãos, tais como a medula óssea, sangue, fígado e baço. Os macrófagos são amplamente distribuídos ao redor do corpo, onde elas nomeadamente participar inata e adaptativa respostas imunes para controlar infecções e claras. Em adição ao seu papel na defesa do hospedeiro, os macrófagos desempenham também um papel importante na cicatrização de feridas e na manutenção da homeostase dos tecidos 1,2. Além disso, os macrófagos não só são importantes para a função imune, mas também participar activamente na homeostase do ferro 3. No corpo, cerca de 80% do ferro está presente na hemoglobina dentro de eritrócitos, que quando senescentes são fagocitadas por macrófagos 4. Diariamente, estes macrófagos reciclar 25 mg de ferro derivados do eritrócito e fornecer o seu transporte para o plasma 5. Além disso, durante a infecção e inflamação, os macrófagos pró-inflamatórias seqüestrar ferro sérico para reduzir availabili ferroty a patógenos, em ambos os níveis sistêmicos e locais 6-8. Assim, os estudos mostraram que os macrófagos e principalmente hepatócitos produzir um péptido antimicrobiano denominado hepcidina que é considerado o principal regulador do metabolismo do ferro 9, 10. A hepcidina é aumentado principalmente por estímulos inflamatórios e é parcialmente responsável pelo seqüestro de ferro em macrófagos em cima inflamação crônica 11-13. Como expressão da hepcidina em macrófagos não é muito bem compreendido, estudamos o possível papel dos receptores Toll-like (TLR) neste regulamento. Os TLRs são encontrados principalmente em macrófagos e desempenham um papel central na sua activação. Além disso, o LPS hepcidina expressão induzida no fígado é dependente de TLR4 13. Portanto, para executar o nosso estudo, foi utilizado um método com base no isolamento de macrófagos peritoneais murinos. Linhas celulares de macrófagos são amplamente utilizados em parafuso prisioneiro de macrófagoss; no entanto, a cultura prolongada pode provocar perda de gene e diminuiu funções imunológicas nestas linhas celulares. Assim, o isolamento de macrófagos da cavidade peritoneal é crucial. A cavidade peritoneal do rato apresenta um local ideal para a colheita macrófagos 13-15. Isolado macrófagos peritoneais de murino são convenientes para diversos estudos em relação à sua função imunológica. No entanto, o número de macrófagos do peritoneu é insuficiente para estudos extensivos e é estimado em cerca de 1 x 10 6 macrófagos por rato. Assim, para aumentar a produção de macrófagos, provocando um agente de estéril tal como tioglicolato foi injectada na cavidade peritoneal anterior à colheita de células. Após a injecção de tioglicolato, o rendimento de macrófagos por rato foi aumentada em 10 vezes. Apesar do aumento na produção de macrófagos, tioglicolato de Brewer médias atos como um irritante que induz uma resposta inflamatória, resultando no recrutamento de macrófagos, que may mas não é necessário afectar a expressão do gene. Assim, um grupo de controlo que consiste em macrófagos não-tratados devem ser incluídos em cada experiência. Nas nossas mãos, hepcidina expressão que é altamente estimulada pela inflamação não foi detectada em tioglicolato não-tratados induziu macrófagos peritoneais. Além disso, estudos têm mostrado que tioglicolato de cerveja recruta numerosos macrófagos, mas não ativá-los 16. Por outro lado, tioglicolato de Brewer eliciada macrófagos mostraram um aumento na enzima lisossómica, mas uma diminuição na morte de microorganismos ingeridos 17. No entanto, a capacidade fagocitária não foi afetado quando comparado com os macrófagos não provocou 16. Uma vez cultivadas em placas, os macrófagos peritoneais se tornar aderente, permitindo assim a sua separação a partir de outro tipo de células isoladas da cavidade peritoneal. Subsequentemente, os macrófagos isolados foram estimulados com diferentes agonistas de TLRs.Por fim, o ARNm foi extraído a partir das células cultivadas e a expressão do gene foi analisada utilizando reacção em cadeia da polimerase-transcriptase reversa quantitativo (qRT-PCR).

<table><tbody><tr><td>C57BL/6 mice</td><td>Charles River Laboratories, Inc. (Wilmington, MA, USA)</td><td>475</td><td></td></tr><tr><td>Thioglycollate</td><td>Sigma-Aldrich, (St. Louis, MO)</td><td>19032-500G</td><td></td></tr><tr><td>70% ethanol</td><td></td><td></td><td></td></tr><tr><td>10% sodium pentobarbital (Used for mice anesthesia (80 mg/kg, i.p.))</td><td></td><td></td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s phosphate-buffered saline (DPBS), placed on ice and will serve to harvest macrophages</td><td>WISENT INC Canada (QC)</td><td>311-425-CL</td><td></td></tr><tr><td>RPMI medium 1640 (Supplement with penicillin, streptomycin, L-glutamine, and 10 % fetal calf serum).</td><td>WISENT INC Canada (QC)</td><td>350-000-CL</td><td></td></tr><tr><td>1 and 5 ml syringes</td><td>BD USA (NJ)</td><td>309659</td><td></td></tr><tr><td>6-well plates</td><td>Corning Incorporated (NY, USA)</td><td>MCT-150-C</td><td></td></tr><tr><td>Bacterial lipoprotein Pam3CSK4 (0.5 mg/ml)</td><td>InvivoGen (San Diego, USA)</td><td>TLRL-pm25</td><td></td></tr><tr><td>Polyionosine&amp;ndash;polycytidylic acid (Poly(I:C)) (10 mg/ml)</td><td>InvivoGen (San Diego, USA)</td><td>TLRL-PIC</td><td></td></tr><tr><td>LPS from Escherichia coli 055:B5 (100 ng/ml)</td><td>InvivoGen (San Diego, USA)</td><td>L2880</td><td></td></tr><tr><td>Purified flagellin from Salmonella typhimurium (100 ng/ml)</td><td>InvivoGen (San Diego, USA)</td><td>TLR-FLIC-10</td><td></td></tr><tr><td>Lipoprotein synthetic FSL1 (100 ng/ml)</td><td>InvivoGen (San Diego, USA)</td><td>TLR-FSL</td><td></td></tr><tr><td>ssRNA derived from the HIV-1 long terminal repeat ssRNA40 (1 &amp;mu;g/ml)</td><td>InvivoGen (San Diego, USA)</td><td>TLR-LRNA-40</td><td></td></tr><tr><td>Type B CpG oligonucleotide ODN1826 (1 &amp;mu;M)</td><td>InvivoGen (San Diego, USA)</td><td>11B16-MM</td><td></td></tr><tr><td>TRIzol</td><td>Invitrogen, (Burlington, ON, Canada)</td><td>15596-026</td><td></td></tr><tr><td>20 G and 23 G needles</td><td>BD USA (NJ)</td><td>305175</td><td></td></tr><tr><td>Scissor</td><td></td><td></td><td></td></tr><tr><td>Forceps</td><td></td><td></td><td></td></tr><tr><td>50 ml conical tubes placed on ice</td><td>Sarstedt (Newton, MA, USA)</td><td>62.547.205</td><td></td></tr><tr><td>Red Blood Cells Lysis Buffer</td><td>Sigma-Aldrich, (St. Louis, MO)</td><td>R7757-100ML</td><td></td></tr><tr><td>Refrigerated centrifuge</td><td></td><td></td><td></td></tr><tr><td>Hemocytometer</td><td></td><td></td><td></td></tr><tr><td>F4/80 antibody</td><td>BIO-RAD ( CA, USA)</td><td>MCA497APC</td><td></td></tr><tr><td>CD16/CD32 antibodies</td><td>Pharmingen, {Mississauga, ON, CA)</td><td>553141</td><td></td></tr><tr><td>Flow cytometer</td><td>Coulter Epics Elite counter, Coulter, (Hialeah, FL,USA)</td><td></td><td></td></tr><tr><td>1.5 ml Eppendorf tubes</td><td>Axygen Scietific (CA,USA)</td><td>3516</td><td></td></tr><tr><td>Chloroform</td><td>Fisher Scientific (ON, Canada)</td><td>UN1888</td><td></td></tr><tr><td>Isopropyl alcohol</td><td>JT Baker (PA, USA)</td><td>70566</td><td></td></tr><tr><td>75% ethanol (in DEPC treated water)</td><td>Commercial Alchohols (QC, Canada)</td><td>17394</td><td></td></tr><tr><td>0.01% diethyl pyrocarbonate (DEPC) treated water (let stand overnight and autoclave)</td><td>Sigma-Aldrich, (St. Louis, MO)</td><td>216.542.8</td><td></td></tr><tr><td>Omniscript RT-PCR system</td><td>Qiagen, (Mississauga, ON, Canada)</td><td>205113</td><td></td></tr><tr><td>Rotor Gene 3000</td><td>Montreal Biotech, (Kirkland, QC, Canada)</td><td></td><td></td></tr><tr><td>QuantiTect SYBR Green I PCR kits</td><td>Qiagen, (Mississauga, ON, Canada)</td><td>204141</td><td></td></tr></tbody></table>

isolation of murine peritoneal macrophages to carry out gene expression analysis upon toll-like receptors stimulation

Durante a infecção e inflamação, os monócitos circulantes na corrente sanguínea e deixar migrar para os tecidos, onde eles diferenciam-se em macrófagos. Os macrófagos expressam receptores de superfície semelhantes a Toll (TLRs) que reconhecem padrões moleculares conservadas através da evolução de uma vasta gama de microorganismos. TLRs desempenhar um papel central na activação de macrófagos, que está geralmente associada com a expressão do gene alteração. Os macrófagos são críticas em muitas doenças e surgiram como alvos atractivos para terapia. No seguinte protocolo, que descrevem um procedimento para isolar macrófagos peritoneais de murino, utilizando meio de tioglicolato de Brewer. Este último irá aumentar a migração dos monócitos para o peritoneu, por conseguinte irá aumentar este rendimento de macrófagos por 10 vezes. Vários estudos têm sido realizados utilizando medula óssea, baço ou peritoneal derivada macrófagos. No entanto, os macrófagos peritoneais foram mostrados para ser mais maduro no isolamento e são mais estáveis ​​no seu funcionaldade e fenótipo. Assim, macrófagos isolados a partir da cavidade peritoneal de murino apresentar uma população de células importante que pode servir em diferentes estudos imunológicos e metabólicos. Uma vez isolados, os macrófagos foram estimulados com diferentes ligandos de TLR e, consequentemente, a expressão do gene foi avaliada.

Nós caracterizado pela primeira vez os macrófagos peritoneais murinos isolados por citometria de fluxo. Para isso, utilizou-se 80 / (F4) anticorpos que reconhecem especificamente marcadores única expressas por macrófagos. Esta caracterização é necessária para determinar a percentagem de macrófagos isolados e para os distinguir entre células obtidas durante o processo de isolamento. Tal como mostrado na (figura 1), a percentagem de células que expressam o antigénio F4 / 80 foi consistentement...

Watch this Scientific Journal Video about Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation at JoVE.com

Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation

We describe here a simple protocol to isolate murine peritoneal macrophages. This procedure is followed by RNA extraction to carry out gene expression analysis upon Toll-like receptors stimulation.

Isolamento de Murino macrófagos peritoniais de Cumprir a análise de expressão gênica Após Toll-like receptors Estimulação

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. ...

isolation-murine-peritoneal-macrophages-to-carry-out-gene-expression

Research

JoVE Journal

Immunology and Infection

36.2K visualizações.  Institut du cancer de Montréal. We describe here a simple protocol to isolate murine peritoneal macrophages. This procedure is followed by RNA extraction to carry out gene expression analysis upon Toll-like receptors stimulation.

Vídeo: Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation

Isolation of Mouse Peritoneal Cavity Cells

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal tract and other viscera. It harbors a number of immune cells including macrophages, B cells and T cells. The presence of a high number of na&iuml;ve macrophages in the peritoneal cavity makes it a preferred site for the collection of na&iuml;ve tissue resident macrophages (1). The peritoneal cavity is also important to the study of B cells because of the presence of a unique peritoneal cavity-resident B cell subset known as B1 cells in addition to conventional B2 cells. B1 cells are subdivided into B1a and B1b cells, which can be distinguished by the surface expression of CD11b and CD5. B1 cells are an important source of natural IgM providing early protection from a variety of pathogens (2-4). These cells are autoreactive in nature (5), but how they are controlled to prevent autoimmunity is still not understood completely. On the contrary, CD5+ B1a cells possess some regulatory properties by virtue of their IL-10 producing capacity (6). Therefore, peritoneal cavity B1 cells are an interesting cell population to study because of their diverse function and many unaddressed questions associated with their development and regulation. The isolation of peritoneal cavity resident immune cells is tricky because of the lack of a defined structure inside the peritoneal cavity. Our protocol will describe a procedure for obtaining viable immune cells from the peritoneal cavity of mice, which then can be used for phenotypic analysis by flow cytometry and for different biochemical and immunological assays.

The peritoneal cavity in mammals contains different immune cell populations crucial for innate immune responses. An efficient isolation method is required for biochemical and functional analyses of these cells. Here we provide a comprehensive method for the isolation of peritoneal cavity cells in the mouse.

Isolamento de células de camundongo Peritoneal Cavity

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal ...

Imunologia e Infecção

A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although the isolation methods of liver macrophages have been well-described4-6, most of these methods require sophisticated equipment, such as a centrifugal elutriator and technical skills. Here, we provide a novel method to obtain liver macrophages in sufficient number and purity from mixed primary cultures of adult rat liver cells, as schematically illustrated in Figure 1.
After dissociation of the liver cells by two-step perfusion method7,8,a fraction mostly composed of parenchymal hepatocytes is prepared and seeded into T75 tissue culture flasks with culture medium composed of DMEM and 10% FCS.Parenchymal hepatocytes lose the epithelial cell morphology within a few days in culture, degenerate or transform into fibroblast-like cells (Figure 2). As the culture proceeds, around day 6, phase contrast-bright, round macrophage-like cells start to proliferate on the fibroblastic cell sheet (Figure 2). The growth of the macrophage-like cells continue and reach to maximum levels around day 12, covering the cell sheet on the flask surface. By shaking of the culture flasks, macrophages are readily suspended into the culture medium. Subsequent transfer and short incubation in plastic dishes result in selective adhesion of macrophages(Figure 3), where as other contaminating cells remain suspended. After several rinses with PBS, attached macrophages are harvested. More than 106 cells can be harvested repeatedly from the same T75 tissue culture flask at two to three day intervals for more than two weeks(Figure 3).The purities of the isolated macrophages were 95 to 99%, as evaluated by flow cytometry or immunocytochemistry with rat macrophage-specific antibodies (Figure 4).The isolated cells show active phagocytosis of polystylene beads (Figure 5), proliferative response to recombinant GM-CSF, secretion of inflammatory/anti-inflammatory cytokines upon stimulation with LPS, and formation of multinucleated giant cells9.
In conclusion, we provide a simple and efficient method to obtain liver macrophages in sufficient number and purity without complex equipment and skills.This method might be applicable to other mammalian species.

A novel method to obtain macrophages from primary culture of rat liver cells is described. This method utilizes the proliferation of macrophages in the culture, followed by shaking of culture flasks and purification by selective attachment to plastic dishes. This technique efficiently provides liver macrophages without complex equipment and skills.

Um método simples e eficiente para isolar Macrófagos de Mixed culturas primárias de células do fígado Adulto

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although ...

Studying the Effects of Tumor-Secreted Paracrine Ligands on Macrophage Activation using Co-Culture with Permeable Membrane Supports

Tumor-derived paracrine signaling is an overlooked component of local immunosuppression and can lead to a permissive environment for continued cancer growth and metastasis. Paracrine signals can involve cell-cell contact between different cell types, such as PD-L1 expressed on the surface of tumors interacting directly with PD-1 on the surface of T cells, or the secretion of ligands by a tumor cell to affect an immune cell. Here we describe a co-culture method to interrogate the effects of tumor-secreted ligands on immune cell (macrophage) activation. This straightforward procedure utilizes commercially available 0.4 &#181;m polycarbonate membrane permeable supports and standard tissue culture plates. In the process described, macrophages are cultured in the upper chamber and tumor cells in the lower chamber. The presence of the 0.4 &#181;m barrier allows for the study of intercellular signaling without the confounding variable of physical contact, because the two cell types share the same medium and exposure to paracrine ligands. This approach can be combined with others, such as genetic alterations of the macrophage (e.g., isolation from genetic knock-out mice) or tumor (e.g., CRISPR-mediated alterations) to study the role of specific secreted factors and receptors. The approach also lends itself to standard molecular biological analyses such as quantitative reverse transcription polymerase chain reaction (qRT-PCR) or Western blot analysis, without the need for flow sorting to separate the two cell populations. Enzyme-linked immunosorbent assays (ELISAs) can similarly be utilized to measure secreted ligands to better understand the dynamic interaction of cell signaling in the multiple cell type context. Duration of co-culture can also be varied for the study of temporally regulated events. This co-culture method is a robust tool that facilitates the study of tumor-secreted signals in the immune context.

Here, we present a method using permeable membrane supports to facilitate the study of non-contact paracrine signaling used by tumor cells to suppress the immune response. The system is amenable to studying the role of tumor-secreted factors in dampening macrophage activation.

Estudando os efeitos dos ligantes paracrinos tumorais na ativação do macrófago usando a co-cultura com suportes permeáveis da membrana

Tumor-derived paracrine signaling is an overlooked component of local immunosuppression and can lead to a permissive environment for continued cancer ...

Pesquisa do câncer

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

Usando RNA-interferência para investigar a imunitário inatas de resposta em macrófagos

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

Isolation of Human Monocytes by Double Gradient Centrifugation and Their Differentiation to Macrophages in Teflon-coated Cell Culture Bags

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to understand the underlying mechanisms of these diseases. We therefore present a straightforward protocol for the isolation of human monocytes from buffy coats, followed by a differentiation procedure which results in high macrophage yields. The technique relies mostly on commonly available lab equipment and thus provides a cost and time effective way to obtain large quantities of human macrophages. Briefly, buffy coats from healthy blood donors are subjected to a double density gradient centrifugation to harvest monocytes from the peripheral blood. These monocytes are then cultured in fluorinated ethylene propylene (FEP) Teflon-coated cell culture bags in the presence of macrophage colony-stimulating factor (M-CSF). The differentiated macrophages can be easily harvested and used for subsequent studies and functional assays. Important methods for quality control and validation of the isolation and differentiation steps will be highlighted within the protocol. In summary, the protocol described here enables scientists to routinely and reproducibly isolate human macrophages without the need for cost intensive tools. Furthermore, disease models can be studied in a syngeneic human system circumventing the use of murine macrophages.

We present a simple and efficient protocol for the generation of human macrophages. Buffy coats are processed by double density gradient centrifugation and isolated monocytes are then differentiated to macrophages in Teflon-coated cell culture bags. This maximizes macrophage yields and facilitates cell harvesting for subsequent experiments.

Isolamento de monócitos humanos por duas vezes centrifugação em gradiente e sua diferenciação de macrófagos em revestidos de teflon Cultura de Células Bags

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to ...

Assessment of Antibody-based Drugs Effects on Murine Bone Marrow and Peritoneal Macrophage Activation

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages are equally important in turning off inflammatory responses. We have shown that macrophages stimulated with intravenous immunoglobulin (IVIg) can produce high amounts of the anti-inflammatory cytokine, interleukin 10 (IL-10), and low levels of pro-inflammatory cytokines in response to bacterial lipopolysaccharides (LPS). IVIg is a polyvalent antibody, primarily immunoglobulin Gs (IgGs), pooled from the plasma of more than 1,000 blood donors. It is used to supplement antibodies in patients with immune deficiencies or to suppress immune responses in patients with autoimmune or inflammatory conditions. Infliximab, a therapeutic anti-tumor necrosis factor alpha (TNF&#945;) antibody, has also been shown to activate macrophages to produce IL-10 in response to inflammatory stimuli. IVIg and other antibody-based biologics can be tested to determine their effects on macrophage activation. This paper describes methods for derivation, stimulation, and assessment of murine bone marrow macrophages activated by antibodies in vitro and murine peritoneal macrophages activated with antibodies in vivo. Finally, we demonstrate the use of western blotting to determine the contribution of specific cell signaling pathways to anti-inflammatory macrophage activity. These protocols can be used with genetically modified mice, to determine the effect of a specific protein(s) on anti-inflammatory macrophage activation. These techniques can also be used to assess whether specific biologics may act by changing macrophages to an IL-10-producing anti-inflammatory activation state that reduces inflammatory responses in vivo. This can provide information on the role of macrophage activation in the efficacy of biologics during disease models in mice, and provide insight into a potential new mechanism of action in people. Conversely, this may caution against the use of specific antibody-based biologics to treat infectious disease, particularly if macrophages play an important role in host defense against that infection.

Antibody-based drugs have revolutionized treatment for inflammatory diseases. In addition to having direct effects on specific targets, antibodies can activate macrophages to become anti-inflammatory. This protocol describes how anti-inflammatory macrophage activation can be assessed in vitro, using mouse bone marrow macrophages, and in vivo, using peritoneal macrophages.

Avaliação dos efeitos de drogas baseados em anticorpo murino da medula óssea e ativação de macrófagos peritoneais

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages ...

Screening Bioactive Nanoparticles in Phagocytic Immune Cells for Inhibitors of Toll-like Receptor Signaling

Pharmacological regulation of Toll-like receptor (TLR) responses holds great promise in the treatment of many inflammatory diseases. However, there have been limited compounds available so far to attenuate TLR signaling and there have been no clinically approved TLR inhibitors (except the anti-malarial drug hydroxychloroquine) in clinical use. In light of rapid advances in nanotechnology, manipulation of immune responsiveness using nano-devices may provide a new strategy to treat these diseases. Herein, we present a high throughput screening method for quickly identifying novel bioactive nanoparticles that inhibit TLR signaling in phagocytic immune cells. This screening platform is built on THP-1 cell-based reporter cells with colorimetric and luciferase assays. The reporter cells are engineered from the human THP-1 monocytic cell line by stable integration of two inducible reporter constructs. One expresses a secreted embryonic alkaline phosphatase (SEAP) gene under the control of a promoter inducible by the transcription factors NF-&#954;B and AP-1, and the other expresses a secreted luciferase reporter gene under the control of promoters inducible by interferon regulatory factors (IRFs).Upon TLR stimulation, the reporter cells activate transcription factors and subsequently produce SEAP and/or luciferase, which can be detected using their corresponding substrate reagents. Using a library of peptide-gold nanoparticle (GNP) hybrids established in our previous studies as an example, we identified one peptide-GNP hybrid that could effectively inhibit the two arms of TLR4 signaling cascade triggered by its prototypical ligand, lipopolysaccharide (LPS). The findings were validated by standard biochemical techniques including immunoblotting. Further analysis established that this lead hybrid had a broad inhibitory spectrum, acting on multiple TLR pathways, including TLR2, 3, 4, and 5. This experimental approach allows a rapid assessment of whether a nanoparticle (or other therapeutic compounds) can modulate specific TLR signaling in phagocytic immune cells.

Toll-like receptor (TLR) signaling plays an important role in the pathophysiology of many human inflammatory diseases, and regulating TLR responses by bioactive nanoparticles is anticipated to be beneficial in many inflammatory conditions. THP-1 cell-based reporter cells provide a versatile and robust screening platform for identifying novel inhibitors of TLR signaling.

Triagem de Nanopartículas Bioativas em Células Imunes Fagocíticas para Inibidores da Sinalização de Receptores Toll-like

Pharmacological regulation of Toll-like receptor (TLR) responses holds great promise in the treatment of many inflammatory diseases. However, there have ...

Medicina

Experimental Analysis of Apoptotic Thymocyte Engulfment by Macrophages

Cell apoptosis is a natural process and plays a critical role in embryonic development, homeostatic regulation, immune tolerance induction, and resolution of inflammation. Accumulation of apoptotic debris in the body may trigger chronic inflammatory responses that lead to systemic autoimmune diseases over time. Impaired apoptotic cell clearance has been implicated in a variety of autoimmune diseases. Apoptotic clearance is a complex process rarely detected under physiological conditions. It involves abundant surface receptors and signaling molecules. Studying the process of apoptotic cell clearance provides insightful molecular mechanisms and subsequent biological responses, which may lead to the development of new therapeutics. Here, we describe protocols for the induction of apoptotic thymocytes, the preparation of peritoneal macrophages, and the analysis of apoptotic cell clearance by flow cytometry and microscopy. All cells will undergo apoptosis at a certain stage, and many residential and circulating cells can uptake apoptotic debris. Therefore, the protocol described here can be used in many applications to characterize apoptotic cell binding and ingestion by many other cell types.

Here, we present a protocol to prepare apoptotic thymocytes and peritoneal macrophages and analyze the efficiency of efferocytosis and the specific inhibitor-mediated blocking of apoptotic thymocytes engulfment. This protocol has a broad application in cell-mediated clearance of other particles including artificial beads and bacteria.

Análise experimental do engulfment do Thymocyte de apoptotic por macrófagos

Cell apoptosis is a natural process and plays a critical role in embryonic development, homeostatic regulation, immune tolerance induction, and resolution ...

Isolation and Culture of Bone Marrow-Derived Macrophages from Mice

Macrophages have important effector functions in homeostasis and inflammation. These cells are present in every tissue in the body and have the important ability to change their profile according to the stimuli present in the microenvironment. Cytokines can profoundly affect macrophage physiology, especially IFN-&#947; and interleukin 4, generating M1 and M2 types respectively. Because of the versatility of these cells, the production of a population of bone marrow-derived macrophages can be a basic step in many experimental models of cell biology. The aim of this protocol is to help researchers in the isolation and culture of macrophages derived from bone marrow progenitors. Bone marrow progenitors from pathogen-free C57BL/6 mice are transformed into macrophages upon exposure to macrophage colony-stimulating factor (M-CSF) that, in this protocol, is obtained from the supernatant of the murine fibroblast lineage L-929. After incubation, mature macrophages are available for use from the 7th to the 10th day. A single animal can be the source of approximately 2 x 107 macrophages. Therefore, it is an ideal protocol for obtaining large amounts of primary macrophages using basic methods of cell culture.

The present protocol describes the isolation and culture of bone marrow-derived macrophages from mice.

Isolamento e cultura de macrófagos derivados da medula óssea de camundongos

Macrophages have important effector functions in homeostasis and inflammation. These cells are present in every tissue in the body and have the important ...

Analysis of Efferocytosis of Apoptotic Thymocytes by Peritoneal Macrophages

Source: Zhen, Y. et al., <a href="https://app.jove.com/v/59731">Experimental Analysis of Apoptotic Thymocyte Engulfment by Macrophages.</a> J. Vis. Exp. (2019)
This video demonstrates a procedure for analyzing peritoneal macrophage-mediated efferocytosis of apoptotic mouse thymocytes by fluorescence microscopy.

Análise da Eferocitose de Timócitos Apoptóticos por Macrófagos Peritoneais

Source: Zhen, Y. et al., Experimental Analysis of Apoptotic Thymocyte Engulfment by Macrophages. J. Vis. Exp. (2019)
This video demonstrates a procedure ...