Name: 视频: 响应抗体激活小鼠骨髓来源的巨噬细胞 - 概念
Uploaded: 2023-08-31T09:44:29.000+00:00
Duration: 2 min 3 s
Description: 151 次观看. 来源:Kozicky， L. et al.， 基于抗体的药物对小鼠骨髓和腹膜巨噬细胞活化影响的评估。J. Vis. Exp. （2017 年） 在本视频中，我们演示了骨髓衍生的巨噬细胞响应细菌脂多糖和静脉注射免疫球蛋白的激活。

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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Bone Marrow Macrophage Derivation and Activation with Antibodies
<ol>
	<li>Perform euthanasia using CO2 asphyxiation.
	<ol>
		<li>Place the mouse in the induction chamber. Euthanize the mouse with 5% isoflurane anesthesia, for 60 - 90 s, until immobile and breathing is deep and slow.</li>
		<li>Turn off isoflurane anesthesia and administer 6 - 8 L/min of CO2 until the mouse has stopped breathing. Leave the mouse with CO2 on for at least another 5 min. Verify that there is no longer a heartbeat or respiration. Perform a cervical dislocation to ensure death. 
		NOTE: 8 - 12 weeks-old mice provide the highest yield of macrophages.</li>
	</ol>
	</li>
	<li>Spray mouse legs with 70% ethanol and pin them with arms and legs stretched out in the supine position over a foam board. With scissors and forceps to hold the skin, make a shallow cut (0.2 cm) to remove the skin and fur from the surface of each of the hind legs. 
	NOTE: Perform this procedure and all tissue culture manipulations in a sterile hood.</li>
	<li>Trim muscle to make tibias and femurs visible. Remove the tibias and femurs, by cutting the bone and muscles just below the hip joint and above the ankles. Trim as much muscle off as possible.</li>
	<li>Spray bones with 70% ethanol and wait 1 min for it to evaporate, then place them in a 6-well plate of bone flush medium (Iscove&#39;s modified Dulbecco&#39;s medium (IMDM), 10% fetal bovine serum (FBS)).</li>
	<li>Trim the ends of the bones by cutting 0.1 cm of bone off the ankle and top of the femur so that the bone cavity is exposed. Ensure that the marrow is visible and a 26-gauge needle can be placed in the bone cavity.</li>
	<li>Cut the bones and muscles to separate the tibias and femurs from the knee joints. Insert a 26-gauge needle attached to a 10 mL syringe into the cavity. Flush the bone marrow into a 50 mL conical tube with 5 mL of bone flush medium. Flush two tibias and two femurs, from one mouse, into each 50 mL tube.</li>
	<li>Pipette up and down several times or vortex the marrow at a slow speed to gently disperse clumps. Strings of marrow should be broken up into small (less than 0.5 mm) flecks of marrow. Fill the conical tube to 50 mL with the bone flush medium. Pipette contents of the conical tube into a 75 cm2 tissue culture-treated flask, and incubate at 37 &#176;C, 5% CO2 for 1 h. 
	NOTE: Mature macrophages and mesenchymal cells will become adherent to the flask and be discarded, while the desired hematopoietic progenitors remain in suspension.</li>
	<li>Pipette the medium with hematopoietic progenitors into a 50 mL conical tube. Centrifuge the tube at 300 x g at room temperature for 5 min. Discard the supernatant and resuspend the pellet in 5 mL of macrophage colony-stimulating factor (MCSF) culture medium (IMDM, 10% FBS, 5 ng/mL MCSF, 100 U/mL penicillin/streptomycin, and 150 &#956;M monothioglycerol (MTG)).</li>
	<li>Count cells using a hemocytometer. Add medium to resuspend cells to a concentration of 0.5 x 106 cells/mL, 1.5 x 107 cells/flask in 30 mL of medium and pipette up and down gently. Pipette 30 mL of suspension into a new 75 cm2 tissue culture-treated flask.</li>
	<li>On day 4 and day 7 after initial culture in step 1.9, verify that the cells are adherent and slightly branched using an inverted phase contrast bright field microscope. Discard the medium containing the non-adherent cells. Wash the cells with IMDM one time and add 30 mL of MCSF culture medium.</li>
	<li>When the cells are mature by day 10 (&#62;95% positive for F4/80 and Mac-1), verify again that the cells are adherent and slightly.</li>
	<li>To plate cells for stimulation, remove the culture medium from the flask and add 8 mL of enzyme-free, EDTA-based cell dissociation buffer (Table of Materials). Incubate the cells for 5 min at 37 &#176;C, 5% CO2.</li>
	<li>Scrape cells gently, with a small amount of pressure, using a sterile cell scraper. Check in the microscope that cells have detached from the flask surface. Pipette dissociated cells into a 50 mL conical tube. Rinse the flask 3 times with 5 mL of IMDM and pool the rinse solution with the cells that were harvested. Centrifuge cells at 300 x g at room temperature for 5 min.</li>
	<li>Resuspend the cell pellet in 3 mL of MCSF culture medium per 50 mL conical tube. Count viable cells using a hemocytometer. Resuspend cells at a concentration of 1 x 106 cells/mL and plate 100 &#956;L of cell suspension (1 x 105 cells/well) per well of a 96-well tissue culture treated, flat bottom plate. 
	NOTE: Bones from one mouse will generate enough cells for 100 wells. If desired, 1 mL of cells can be plated in a 6-well plate (tissue culture treated, flat bottom). A larger number of cells (1 x 106 cells) may be useful for western blot analyses.</li>
	<li>Once adherent, stimulate duplicate or triplicate wells each with 10 ng/mL of LPS in IMDM, 30 mg/mL of IVIg, or IVIg + LPS. Doses of LPS or IVIg can be titrated to optimize responses. Leave duplicate or triplicate wells as unstimulated controls. Incubate them for 24 h (37 &#176;C, 5% CO2). 
	NOTE: Re-plated cells should adhere to tissue culture wells within 1 h.</li>
	<li>Collect cell supernatants from each well into individual 1.7 mL microcentrifuge tubes and remove any particulate matter by spinning at 10,000 x g for 5 min.</li>
	<li>Remove the cell supernatant and avoid disturbing the pellet. Place the clarified cell supernatant in a sterile microcentrifuge tube. 
	NOTE: Cell supernatants can be assayed for cytokines, IL-10, and cytokine subunit, IL-12/23p40, by enzyme-linked immunosorbent assay (ELISA) immediately or stored at -80 &#176;C long term. Follow the ELISA protocol from a commercially available kit (Table of Materials). Other pro-inflammatory cytokines can be assayed, such as IL-6 and TNF, as they are also reduced by co-treatment with IVIg + LPS stimulation compared to stimulation with LPS alone. After stimulation, adherent cells can be prepared for techniques such as western blotting or quantitative polymerase chain reaction (Q-PCR).</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Iscove&#39;s modified Dulbecco&#39;s medium (IMDM)</td>
			<td>Life technologies</td>
			<td>12440053</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Life technologies</td>
			<td>12483-020</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant murine macrophage colony-stimulating factor (MCSF)</td>
			<td>Stemcell technologies</td>
			<td>78059</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>Life technologies</td>
			<td>15140148</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell dissociation buffer</td>
			<td>Life technologies</td>
			<td>13150016</td>
			<td>Enzyme-Free, Hanks&#39;s-based, EDTA</td>
		</tr>
		<tr>
			<td>Lipopolysaccharide (LPS)</td>
			<td>Sigma aldrich</td>
			<td>L 4516</td>
			<td>From E. coli 0127:B8</td>
		</tr>
		<tr>
			<td>IVIg (Gammunex)</td>
			<td>Grifols</td>
			<td>Received from BC Children&#39;s Hospital, Transfusion Medicine</td>
			<td></td>
		</tr>
		<tr>
			<td>IVIg (Gammagard liquid)</td>
			<td>Baxter Healthcare Corporation</td>
			<td>Received from BC Children&#39;s Hospital, Transfusion Medicine</td>
			<td></td>
		</tr>
		<tr>
			<td>IVIg (Octagam)</td>
			<td>Octapharma</td>
			<td>Received from BC Children&#39;s Hospital, Transfusion Medicine</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS) (sterile), pH 7.4</td>
			<td>Life technologies</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse IL-10 ELISA</td>
			<td>BD biosciences</td>
			<td>555252</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse IL-12/23p40 ELISA</td>
			<td>BD biosciences</td>
			<td>555165</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL conical tube</td>
			<td>BD biosciences</td>
			<td>352096</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical tube</td>
			<td>BD biosciences</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tube (1.7 mL)</td>
			<td>Diamed</td>
			<td>SPE155-N</td>
			<td></td>
		</tr>
		<tr>
			<td>75 cm2 tissue culture treated flask</td>
			<td>BD biosciences</td>
			<td>353136</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell scraper</td>
			<td>BD biosciences</td>
			<td>353085</td>
			<td></td>
		</tr>
	</tbody>
</table>

activation of mouse bone marrow-derived macrophages in response to antibodies

Source: Kozicky, L. et al., <a href="https://app.jove.com/v/56689">Assessment of Antibody-based Drugs Effects on Murine Bone Marrow and Peritoneal Macrophage Activation.</a> J. Vis. Exp. (2017)
In this video, we demonstrate the activation of bone marrow-derived macrophages in response to bacterial lipopolysaccharide and intravenous immunoglobulin.

Activation of Mouse Bone Marrow-Derived Macrophages in Response to Antibodies

Source: Kozicky, L. et al., Assessment of Antibody-based Drugs Effects on Murine Bone Marrow and Peritoneal Macrophage Activation. J. Vis. Exp. (2017)
In ...

Following pathogen exposure, macrophages undergo activation and release pro-inflammatory and anti-inflammatory cytokines, coordinating the immune system response.
To study bone marrow-derived macrophage activation in vitro, transfer an isolated mouse bone marrow cell suspension to a culture flask. Briefly incubate.
The mesenchymal cells, monocytes, and mature macrophages adhere to the flask surface while the hematopoietic progenitors remain in suspension.
Collect the media containing hematopoietic progenitors and centrifuge. Transfer the cells resuspended in macrophage colony-stimulating factor, M-CSF, media to a culture flask.
M-CSF causes the myeloid progenitor subpopulation to differentiate into macrophages, which appear branched and adhere to the flask.
Discard the non-adherent cell-containing media. Add M-CSF media, and incubate. Once the macrophages reach their fully functional state, harvest them. Centrifuge.
Plate macrophages resuspended in M-CSF media into multi-well plate wells. Following macrophage adherence to the wells, add human polyclonal immunoglobulins and bacterial lipopolysaccharide, LPS, a toxin.
Immunoglobulins bind to specific FC&#947; receptors, while LPS binds to Toll-like receptors on macrophages. This co-stimulation drives anti-inflammatory macrophage activation via downstream signaling cascades, causing the release of high levels of anti-inflammatory cytokines and low levels of pro-inflammatory cytokines.
Collect the cytokine-containing media for further analysis to confirm macrophage activation.

activation-mouse-bone-marrow-derived-macrophages-response-to

Research

JoVE Encyclopedia of Experiments

Immunology

Immune Response

Host Defense Mechanism

151 次观看. 来源:Kozicky， L. et al.， 基于抗体的药物对小鼠骨髓和腹膜巨噬细胞活化影响的评估。J. Vis. Exp. （2017 年） 在本视频中，我们演示了骨髓衍生的巨噬细胞响应细菌脂多糖和静脉注射免疫球蛋白的激活。 

视频: 响应抗体激活小鼠骨髓来源的巨噬细胞 - 概念

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.

偏光M1和M2骨髓来源的巨噬细胞的使用实时外流量分析代谢表征

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

科研

JoVE Journal

免疫与感染

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.

代和GM-CSF的鉴定源性肺泡状巨噬细胞和树突状细胞从小鼠骨髓

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. ...

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.

骨髓来源巨噬细胞的分离和 体外 培养用于NO-氧化还原生物学研究

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

Activation of Mouse Bone Marrow-Derived Macrophages in Response to Antibodies

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Activation of Mouse Bone Marrow-Derived Macrophages in Response to Antibodies