Diese Arbeit wurde von der American Heart Association (BGIA 7850037 Dr. Beiyan Zhou) unterstützt.

1. Isolation von Knochenmarkzellen <ol><li> Isolieren Oberschenkels und des Schienbeins Knochen von 6-8 Wochen alten Mäusen, abspülen Haar und dann aufgeschnitten den Knochen. </li><li> Verwenden Sie eine Nadel 21G und 10-ml-Spritze zu spülen Knochenmark in kaltem PBS +2% hitzeinaktiviertes Fetal Bovine Serum (FBS) (3-5 ml / Maus). </li><li> Pass Knochenmark durch eine 21G Nadel 4-6 mal um die Zellen zu distanzieren. </li><li> Pass Zellen durch eine 70 um Zellsieb zu Zellklumpen, Knochen, Haare und andere Zellen / Gewebe zu entfernen. </li><li> In 3 Volumen von NH 4 Cl-Lösung (0,8% NH 4 Cl-Lösung, Stemcell Technology), und Inkubation auf Eis für 10 min, um rote Blutkörperchen zu entfernen. </li><li> Spin down Zellen bei 500 xg für 5 min bei 4 ° C. </li><li> Zellpellet in kaltem PBS + 2% FBS (20-50 ml, je nach der Menge der Zellen). </li></ol> 2. Induction BMDM Formation <ol><li> Resuspendieren der isolierten Knochenmarkzellen in BMDM Wachstumsmedium (2x10 6 Zellen / ml). </li></ol> BMDM Wachstumsmedium: Iscoves Modified Dulbecco-Medium (IMDM) + 10% FBS + 15% gefiltert (0,2 um) L-929-Zellen (ATCC, CCL-1) Kulturüberstand (mit Monozyten-Kolonie-stimulierender Faktor, M-CSF) oder 10 ng / ml M -CSF. Hinweis: L-929 Zellüberstand enthält M-CSF 18. Um die effektive Aktivität des konditionierten Mediums, 5 X 10 5 sicherzustellen L-929-Zellen in T75 cm 2-Kolben für 6-7 Tage ausgesät, konditionierte Medium gesammelt und durch ein 0,45 um-Filter vor dem Gebrauch übergeben. Aliquots Medium kann sofort verwendet werden oder gespeichert in -80 ° C für 1-2 Monate. <ol start="2"><li> Seed-Zellen in 6 oder 12 Loch-Gewebekulturplatten (je nach Versuchsaufbau) (Corning Costar). </li><li> Ändern frisch BMDM Wachstumsmedium am 3. Tag. </li><li> Am 7. Tag wird die Bildung von reifen BMDM ausgewertet mittels Durchflusszytometrie-Analyseund Fluorophor konjugierten Antikörpern zu erkennen exprimieren CD11b und F4/80. </li></ol> 3. BMDM Polarized Aktivierung <ol><li> Am Tag 7, an die frische Stimulation Medium ändern: für M1 Aktivierung verwenden IMDM mit 10% FBS und 100 ng / ml LPS oder 100 ng / ml LPS mit 50 ng / ml IFN; M2 für Aktivierung verwenden IMDM mit 10% FBS mit 10 ng / ml IL-4 und / oder 10 ng / ml IL-13. </li><li> Sammeln angeregt BMDMs durch Lösen sie aus der Schale mit warmem 0,05% Trypsin, gefolgt vom Waschen der Zellen zweimal mit PBS, enthaltend 10% FBS. </li></ol> Hinweis: Um lösen und suspendieren reifen Makrophagen nach der Differenzierung, 0,05% Trypsin-Lösung (enthaltend 0,48 mM EDTA, Invitrogen) oder 2-5 mM EDTA in Ca-und Mg-freiem PBS oder Hanks-Puffer (HBSS) verwendet werden. Bei der Verwendung von Trypsin Verdauung Medium werden die Zellen bei 37 ° C für weniger als 10 Minuten behandelt, um Verlust zu vermeiden surGesicht Proteine ​​aufgrund über-Verdauung. <ol start="3"><li> Mit Antikörpern, um die Expression von Zelloberflächen-Antigene, einschließlich CD11b, F4/80, CD11c, CD206, CD69, CD80 oder CD86 zu verschiedenen Zeitpunkten mit Standard Durchflusszytometrie Färbeverfahren. </li><li> Bestimmen Expression von Genen charakteristisch aktivierten M1 und M2 Makrophagen einschließlich IL-1β, TNF-α und IL-6 (M1 Aktivierung) oder IL-10, IL-13, arginase1 und PPAR (M2 Aktivierung) mit qRT-PCR. Bestimmen Aktivierung der Zelle Signalwege in der Aktivierung von M1 oder M2 Makrophagen durch Western-Blot-Analyse beteiligt. </li></ol>

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Wir berichten hier über eine einfache und leicht anpassbar in vitro Verfahren zur Aktivierung von Makrophagen aus Knochenmark-Vorläuferzellen abgeleitet induzieren. Dieses Verfahren kann zur Untersuchung der Mechanismen, die für die Polarisation von Makrophagen verwendet werden. Die Reinheit von reifen Makrophagen unter Verwendung dieses Protokolls beträgt 95 bis 99%, und keine zusätzlichen Reinigungsschritte benötigt werden. Um die Funktion bestimmter Gene von Interessen im Rahmen der Makrophagen-Polaris...

Im Unterschied zu klassischen Entzündungsreaktionen, Makrophagen, die Gewebe infiltrieren zeigen oft polarisiert Aktivierungsstatus, die eine entscheidende Rolle bei der Regulierung Wirtsgewebe physiologischen Funktionen 1-8 spielt. Nach Stimulation kann Makrophagenaktivierung in classic (M1) und alternativen (M2) Aktivierung 2, 4, 9 sortiert werden. M1 Makrophagenaktivierung hängt von Toll-like Rezeptoren (TLR) und Aktivierung von nuclear factor kappa B (NFKB) / c-Jun N-terminal kinase 1 (JNK1), was zur Produktion von inflammatorischen Zytokinen wie TNF-α und IL- 1β und Aktivierung von iNOS, die zu einer erhöhten Produktion von reaktiven Sauerstoffspezies wie Nitrid-Oxid (NO) 10, 11. Im Gegensatz dazu führt M2 Makrophagen-Aktivierung rekrutiert PPAR, PPAR oder IL-4-STAT6 Wege, um alternative, entzündungshemmende (M2), die mit Aktivierung Hochregulation von Mannose-Rezeptor CD206 zugeordnet ist, und Arginase 1 (Arg1) 6, 12 - 14 &lt;/ Sup&gt;. Knochenmark abgeleiteten Makrophagen (BMDM) präsentieren eine ideale in vitro Modell, um die Mechanismen, die die Polarisation des aktivierten Makrophagen 15 zu verstehen. Insbesondere kann die Aktivierung von Makrophagen M1 durch Lipopolysaccharide (LPS) Stimulation induziert werden, während Polarisation M2 Makrophagen IL-4 und / oder IL-13 induziert werden kann. Ältere Knochenmark stammende Makrophagen und aktivierten Makrophagen durch Durchflusszytometrie-Analyse auf die Expression von Oberflächen-Antigenen, einschließlich CD11b, F4/80, CD11c, CD206, CD69, CD80 und CD86 9, 16, 17 identifiziert werden. Darüber hinaus können Änderungen in der Produktion von Zytokinen und Zellsignalwege mit Makrophagen Polarisationsrichtung zugeordnet ist, durch quantitative RT-PCR und Western Blot, jeweils gemessen werden. Zusammenfassend kann Knochenmark der Maus Makrophagen als relevantes Modell Makrophagen Polarisation in-vitro-Studie dienen.

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>IMDM</td>
 <td>Thermo Scientific</td>
 <td>SH30259.01</td>
 <td></td>
 </tr>
 <tr>
 <td>Fetal bovine serum</td>
 <td>Invitrogen</td>
 <td>10438-026</td>
 <td></td>
 </tr>
 <tr>
 <td>Murine GM-CSF</td>
 <td>PeproTech</td>
 <td> 315-03</td>
 <td></td>
 </tr>
 <tr>
 <td>NH4Cl</td>
 <td>StemCell Technologies</td>
 <td>7850</td>
 <td></td>
 </tr>
 <tr>
 <td>L-929</td>
 <td>ATCC</td>
 <td>CCL-1</td>
 <td></td>
 </tr>
 <tr>
 <td>70 &mu;m cell strainer</td>
 <td>BD Biosciences</td>
 <td>352350</td>
 <td></td>
 </tr>
 <tr>
 <td>10 x PBS</td>
 <td>Thermo Scientific</td>
 <td>AP-9009-10</td>
 <td></td>
 </tr>
 <tr>
 <td>Anti-mouse CD11b-APC</td>
 <td>eBioscience</td>
 <td>17-0112-81</td>
 <td></td>
 </tr>
 <tr>
 <td>Anti-mouse F4/80-FITC</td>
 <td>eBioscience</td>
 <td>11-4801-81</td>
 <td></td>
 </tr>
 <tr>
 <td>Anti-mouse CD69-PE</td>
 <td>eBioscience</td>
 <td>12-0691-81</td>
 <td></td>
 </tr>
 <tr>
 <td>Anti-mouse CD86-PE</td>
 <td>eBioscience</td>
 <td>12-0862-81</td>
 <td></td>
 </tr>
 <tr>
 <td>Propidium Iodine</td>
 <td>Invitrogen</td>
 <td>P3566</td>
 <td></td>
 </tr>
 </tbody>
</table>

investigation of macrophage polarization using bone marrow derived macrophages

Der Artikel beschreibt eine einfache adaptive leicht in vitro Modell zur Makrophagen-Polarisierung zu untersuchen. In Gegenwart von GM-CSF/M-CSF, hämatopoetische Stammzellen sind / Vorläuferzellen aus dem Knochenmark in Monozyten Differenzierung gerichtet, gefolgt von M1 oder M2 Stimulation. Der Aktivierungsstatus kann durch Änderungen in Zelloberflächen-Antigene, Genexpression und Zellsignalwege verfolgt werden.

Eine schematische Beschreibung des BMDM Generation Verfahren vorgestellt (Abbildung 1). Hohe Reinheit von reifen Makrophagen an Tag 7 beobachtet werden, wenn sie 95 bis 99% der CD11b + F4/80 + Zellen (Abbildung 2) zu vertreten. Polarisierte Makrophagen untersucht werden unter Verwendung von Antikörpern gegen CD11b, F4/80, gefolgt CD11c und CD206 mittels Durchflusszytometrie-Analyse. Wie in Abbildung 3 gezeigt, sind M1 Makrophagen erkannt CD11b + CD11c + + F4/80 CD206-Z...

Watch this Scientific Journal Video about Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages at JoVE.com

Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages

Der Artikel beschreibt eine leicht einfach adaptive<em&gt; In vitro</em&gt; Modell Makrophagen Polarisation zu untersuchen. In Gegenwart von GM-CSF/M-CSF, hämatopoetische Stammzellen sind / Vorläuferzellen aus dem Knochenmark in Monozyten Differenzierung gerichtet, gefolgt von M1 oder M2 Stimulation. Der Aktivierungsstatus kann durch Änderungen in Zelloberflächen-Antigene, Genexpression und Zellsignalwege verfolgt werden.

Untersuchung von Makrophagen-Polarisation mit Knochenmark-Makrophagen

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic ...

investigation-macrophage-polarization-using-bone-marrow-derived

Research

JoVE Journal

Immunology and Infection

65.5K Aufrufe.  Texas A&M University. Der Artikel beschreibt eine leicht einfach adaptive In vitro Modell Makrophagen Polarisation zu untersuchen. In Gegenwart von GM-CSF/M-CSF, hämatopoetische Stammzellen sind / Vorläuferzellen aus dem Knochenmark in Monozyten Differenzierung gerichtet, gefolgt von M1 oder M2 Stimulation. Der Aktivierungsstatus kann durch Änderungen in Zelloberflächen-Antigene, Genexpression und Zellsignalwege verfolgt werden.

Serie: Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages

Toxoplasma gondii Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions

Toxoplasma gondii is an obligate intracellular parasite that can invade any nucleated cell of warm-blooded animals. During infection, T. gondii disseminates as a fast replicating form called the tachyzoite. Tachyzoites convert into a slow-growing encysted form called the bradyzoite by a signaling process that is not well characterized. Within animals, bradyzoite cysts are found in the central nervous system and muscle tissue and represent the chronic stage of infection. Conversion to bradyzoites can be simulated in tissue culture by CO2 starvation, using medium with high a pH, or the addition of interferon gamma (IFN&gamma;). Bradyzoites are characterized by the presence of a cyst wall, to which the lectin Dolichos biflorus agglutinin (DBA) binds. Fluorescently labeled DBA is used to visualize the cyst wall in parasites grown in human foreskin fibroblasts (HFFs) that have been exposed to low CO2 and high pH medium. Similarly, parasites residing in murine bone marrow-derived macrophages (BMMs) display a cyst wall detectable by DBA after the BMMs are activated with IFN&gamma; and lipopolysaccharide (LPS). This protocol will demonstrate how to induce conversion of T. gondii to bradyzoites using a high pH growth medium with low CO2 and activation of BMMs. Host cells will be cultured on coverslips, infected with tachyzoites and either activated with addition of IFN&gamma; and LPS (BMMs) or exposed to a high pH growth medium (HFFs) for three days. Upon completion of infections, host cells will be fixed, permeabilized, and blocked. Cyst walls will be visualized using rhodamine DBA with fluorescence microscopy.

Toxoplasma gondii Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions

Toxoplasma gondii converts to a cyst form in response to environmental stresses, which can be mimicked in tissue culture models. This video demonstrates techniques to examine cyst wall formation by activating bone marrow-derived macrophages or changing growth medium pH in fibroblast cells.

Toxoplasma gondii Zystenwand Formation in Activated Knochenmark abgeleiteten Makrophagen und Bradyzoite AGB

Toxoplasma gondii is an obligate intracellular parasite that can invade any nucleated cell of warm-blooded animals. During infection, T. gondii ...

Forschung

Immunologie und Infektion

Bone Marrow-derived Macrophage Production

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because of their powerful microbicidal activities. Macrophages are widely distributed throughout the body and are present in the lymphoid organs, liver, lungs, gastrointestinal tract, central nervous system, bone, and skin. Because of their repartition, they participate in a wide range of physiological and pathological processes. Macrophages are highly versatile cells that are able to recognize microenvironmental alterations and to maintain tissue homeostasis. Numerous pathogens have evolved mechanisms to use macrophages as Trojan horses to survive, replicate in, and infect both humans and animals and to propagate throughout the body. The recent explosion of interest in evolutionary, genetic, and biochemical aspects of host-pathogen interactions has renewed scientific attention regarding macrophages. Here, we describe a procedure to isolate and cultivate macrophages from murine bone marrow that will provide large numbers of macrophages for studying host-pathogen interactions as well as other processes.

Macrophages have long been recognized as a critical component of the innate and adaptive immune responses. The recent explosion of knowledge pertaining to evolutionary, genetic, and biochemical aspects of the interaction between macrophages and microbes has renewed scientific attention to macrophages. This article describes a method to differentiate macrophages from mouse bone marrow.

Knochenmark-abgeleiteten Makrophagen-Produktion

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

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

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

Isolierung und intravenöse Injektion von Maus-Knochenmark stammenden Monozyten

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

Metabolic Characterization of Polarized M1 and M2 Bone Marrow-derived Macrophages Using Real-time Extracellular Flux Analysis

Specific metabolic pathways are increasingly being recognized as critical hallmarks of macrophage subsets. While LPS-induced classically activated M1 or M(LPS) macrophages are pro-inflammatory, IL-4 induces alternative macrophage activation and these so-called M2 or M(IL-4) support resolution of inflammation and wound healing. Recent evidence shows the crucial role of metabolic reprogramming in the regulation of M1 and M2 macrophage polarization. 
In this manuscript, an extracellular flux analyzer is applied to assess the metabolic characteristics of naive, M1 and M2 polarized mouse bone marrow-derived macrophages. This instrument uses pH and oxygen sensors to measure the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), which can be related to glycolytic and mitochondrial oxidative metabolism. As such, both glycolysis and mitochondrial oxidative metabolism can be measured in real-time in one single assay.
Using this technique, we demonstrate here that inflammatory M1 macrophages display enhanced glycolytic metabolism and reduced mitochondrial activity. Conversely, anti-inflammatory M2 macrophages show high mitochondrial oxidative phosphorylation (OXPHOS) and are characterized by an enhanced spare respiratory capacity (SRC). 
The presented functional assay serves as a framework to investigate how particular cytokines, pharmacological compounds, gene knock outs or other interventions affect the macrophage&#8217;s metabolic phenotype and inflammatory status.

Metabolic reprogramming is a characteristic and prerequisite for M1 and M2 macrophage polarization. This manuscript describes an assay for the measurement of fundamental parameters of glycolysis and mitochondrial function in mouse bone marrow-derived macrophages. This tool can be applied to investigate how particular factors affect the macrophage&#8217;s metabolism and phenotype.

Metabolische Charakterisierung Polarized M1 und M2-Knochenmark-Makrophagen über die Echtzeit-Extrazelluläre Flußanalyse

Specific metabolic pathways are increasingly being recognized as critical hallmarks of macrophage subsets. While LPS-induced classically activated M1 or ...

Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. However, they are difficult to isolate in sufficient numbers to study them in detail, therefore, in vitro models have been developed. In vitro cultures of bone marrow-derived macrophages and dendritic cells are well-established and valuable methods for immunological studies. Here, a method for culturing and identifying both DCs and macrophages from a single culture of primary mouse bone marrow cells using the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF) is described. This protocol is based on the established procedure first developed by Lutz et al. in 1999 for bone marrow-derived DCs. The culture is heterogeneous, and MHCII and fluoresceinated hyaluronan (FL-HA) are used to distinguish macrophages from immature and mature DCs. These GM-CSF derived macrophages provide a convenient source of in vitro derived macrophages that closely resemble alveolar macrophages in both phenotype and function.

Bone marrow cells cultured with granulocyte macrophage colony stimulating factor (GM-CSF) generate a heterogeneous culture containing macrophages and dendritic cells (DCs). This method highlights using MHCII and hyaluronan (HA) binding to differentiate macrophages from the DCs in the GM-CSF culture. Macrophages in this culture have many similarities to alveolar macrophages.

Erzeugung und Identifizierung von GM-CSF Abgeleitet Alveolare ähnlichen Makrophagen und dendritische Zellen aus Knochenmark der Maus

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. ...

Culture of Macrophage Colony-stimulating Factor Differentiated Human Monocyte-derived Macrophages

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation of experiments, fresh or frozen monocytes are cultured in flasks for 1 week with M-CSF to induce their differentiation into macrophages. Then, the macrophages can be harvested and seeded into culture wells at required cell densities for carrying out experiments. The use of defined numbers of macrophages rather than defined numbers of monocytes to initiate macrophage cultures for experiments yields macrophage cultures in which the desired cell density can be more consistently attained. Use of cryopreserved monocytes reduces dependency on donor availability and produces more homogeneous macrophage cultures.

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. The protocol utilizes cryopreservation of monocytes coupled with their bulk differentiation into macrophages. Then harvested macrophages can then be seeded into culture wells at required cell densities for carrying out experiments.

Kultur von M-CSF Differentiated Menschliche Monozyten abgeleiteten Makrophagen

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation ...

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.

Bewertung der Antikörper-basierte Medikamente Auswirkungen auf murinen Knochenmark und peritonealen Makrophagen Aktivierung

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

Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote inflammatory and autoinflammatory diseases. Inflammasomes are activated by cytosolic pattern recognition receptors, including members of the NOD-like receptor (NLR) family. These receptors oligomerize upon the detection of microbial or damage-associated stimuli. Subsequent recruitment of the adaptor protein ASC forms a microscopically visible inflammasome complex, which activates caspase-1 through proximity-induced auto-activation. Following the activation, caspase-1 cleaves pro-IL-1&#946; and pro-IL-18, leading to the activation and secretion of these pro-inflammatory cytokines. Caspase-1 also mediates the inflammatory form of cell death termed pyroptosis, which features the loss of membrane integrity and cell lysis. Caspase-1 cleaves gasdermin D, releasing the N-terminal fragment which forms plasma membrane pores, leading to osmotic lysis.
In vitro, the activation of caspase-1 can be determined by labeling bone marrow-derived macrophages with the caspase-1 activity probe FAM-YVAD-FMK and by labeling the cells with antibodies against the adaptor protein ASC. This technique allows the identification of inflammasome formation and caspase-1 activation in individual cells using fluorescence microscopy. Pyroptotic cell death can be detected by measuring the release of cytosolic lactate dehydrogenase into the medium. This procedure is simple, cost effective and performed in a 96-well plate format, allowing adaptation for screening. In this manuscript, we show that activation of the NLRP3 inflammasome by nigericin leads to the co-localization of the adaptor protein ASC and active caspase-1, leading to pyroptosis.

We describe the detection of NLRP3 inflammasome activation on cellular basis using fluorescence microscopy and staining for active caspase-1 and the adaptor, ASC. A lactate dehydrogenase release assay is presented to detect pyroptotic lysis on a population basis. These techniques can be adapted to study many aspects of inflammasome biology.

Erkennung von Inflammasome Aktivierung und Pyroptotic Zelltod in murinen Makrophagen Knochenmark abgeleitet

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote ...

Isolation and In vitro Culture of Bone Marrow-Derived Macrophages for the Study of NO-Redox Biology

Macrophages are derived from hematopoietic progenitor cells throughout the body, are central to inflammatory processes, and participate in innate and adaptive immune responses. In vitro study of macrophages can be undertaken by ex vivo culture from the peritoneum or through differentiation of myeloid bone marrow progenitor cells to form bone marrow-derived macrophages (BMDMs). A common approach to macrophage differentiation from precursors involves the use of conditioned media from L929 cells (LCM). This media is easy to self-produce but suffers from batch variability, and its constituents are undefined. Similarly, Foetal Bovine Serum (FBS) is used to support growth but contains a vast mixture of undefined molecules that may vary between batches. These methods are not adequate for the study of nitric oxide biology and redox mechanisms as they both contain substantial amounts of small molecules that either interfere with redox mechanisms or supplement levels of cofactors, such as tetrahydrobiopterin (BH4), required for the production of NO from inducible nitric oxide synthase (iNOS). In this report, we present an optimized protocol allowing for control of the NO-redox environment by reducing the levels of exogenous biopterin while maintaining conditions suitable for cell growth and differentiation. Tight control of culture media composition helps ensure experimental reproducibility and facilitates accurate interpretation of results. In this protocol, BMDMs were obtained from a GTP cyclohydrolase (GCH)- deficient mouse model. Culture of BMDMs was performed with media containing either (i) conditioned LCM, or (ii) recombinant M-CSF and GM-CSF to produce minimal artifacts while obtaining BH4 and NO-deficient culture conditions - thus allowing for the reproducible study of NO-redox biology and immunometabolism in vitro.

Isolation and In vitro Culture of Bone Marrow-Derived Macrophages for the Study of NO-Redox Biology

This protocol has been established to culture tetrahydrobiopterin (BH4)- and inducible nitric oxide synthase (iNOS)-deficient primary murine macrophages to study NO-redox biology. The study focuses on reducing potential contamination of BH4 and other artifacts found in traditional isolation and culture methods which may confound experimental outcomes and interpretation of results.

Isolierung und In vitro Kultur von Knochenmark-abgeleiteten Makrophagen für das Studium der NO-Redox-Biologie

Macrophages are derived from hematopoietic progenitor cells throughout the body, are central to inflammatory processes, and participate in innate and ...

Economical and Efficient Protocol for Isolating and Culturing Bone Marrow-derived Dendritic Cells from Mice

The demand for dendritic cells (DCs) is gradually increasing as immunology research advances. However, DCs are rare in all tissues. The traditional method for isolating DCs primarily involves inducing bone marrow (BM) differentiation into DCs by injecting large doses (&#62;10 ng/mL) of granulocyte-macrophage colony-stimulating factor/interleukin-4 (GM-CSF/IL-4), making the procedure complex and expensive. In this protocol, using all BM cells cultured in 10 ng/mL GM-CSF/IL-4 medium, after 3-4 half-culture exchanges, up to 2.7 x 107 CD11c+ cells&#160;(DCs) per mouse (two femurs) were harvested with a purity of 80%-95%. After 10 days in culture, the expression of CD11c, CD80, and MHC II increased, whereas the number of cells decreased. The number of cells peaked after 7 days of culture. Moreover, this method only took 10 min to harvest all bone marrow cells, and a high number of DCs were obtained after 1 week of culture.

Here, we present an economical and efficient method to isolate and generate high-purity bone marrow-derived dendritic cells from mice after 7 days of culture with 10 ng/mL GM-CSF/IL-4.

Wirtschaftliches und effizientes Protokoll zur Isolierung und Kultivierung von aus Knochenmark gewonnenen dendritischen Zellen aus Mäusen

The demand for dendritic cells (DCs) is gradually increasing as immunology research advances. However, DCs are rare in all tissues. The traditional method ...