Name: generation and identification of gm-csf derived alveolar-like macrophages and dendritic cells from mouse bone marrow
Uploaded: 2016-06-25T14:00:00
Description: Watch this Scientific Journal Video about Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow at JoVE.com

这项工作是由卫生研究院（CIHR）（批准MOP-119503）和加拿大自然科学与工程理事会（NSERC）的加拿大学院资助。 NSERC还支持夏季助学金为YD和AA YD是由英属哥伦比亚大学（UBC）大学用4年的奖学金奖励支持，AA由CIHR与研究生硕士奖（CGS-M）的支持。我们感谢卡尔文Roskelley与用于生成图2中的图像在显微镜的帮助。我们也承认从UBC动物和流式细胞仪设备的支持。

小鼠按照加拿大理事会关于道德的动物研究动物护理指南由不列颠哥伦比亚省的动物护理委员会的批准大学的程序进行安乐死。 1.获得从小鼠股骨和胫骨单细胞骨髓暂停<ol><li>开关上的生物安全柜（BSC）之前15分钟开始的过程的吹扫柜内空气，并允许空气流稳定，用70％的乙醇，然后清洁/喷雾BSC表面。一切都保持无菌尽可能为协议的全部内容。不切非无菌条件下的骨头。 </li><li>通过在70％?...

<ol>
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在这个手稿，我们提供了从正在从Lutz 等人的6适于单个小鼠骨髓培养产生的GM-CSF衍生的巨噬细胞和DC的方法。 MHCII表达和FL-HA结合未成熟DC和巨噬细胞区分在这种文化中（ 见图3C），而此前已经很难。这一点，与Helft 等 19另一份报告，都表明以前被认为只的DC GM-CSF诱导BMDC文化中的异质性。 Helft 等 19用完全不同的培养条件下产生的BMDCs但也发?...

几个体外培养的方法已被描述，以产生骨髓来源的巨噬细胞（BMDMs），并使用一种或生长因子的组合骨髓衍生的DC（DC培养上清）。 BMDMs可以通过使用细胞巨噬细胞集落刺激因子（M-CSF）或GM-CSF 1,2-培养骨髓细胞生成。为DC培养上清，加入FLT3配体与骨髓培养产生了非粘附经典和浆的DC（CD11c的高 / MHCII 高和CD11c的LO，B220分别+）后的培养3,4- 9天。与此相反，后7至10天在培养与单独的GM-CSF 5,6-产生的非贴壁细胞，GM-CSF和IL-4 7，或GM-CSF和FLT3配体8,9-生成的BMDCs更密切类似的炎症的DC （CD11c的高 ， 高 MHCII +细胞CD11b）10。而这些在体外培养物被用于产生巨噬细胞或区议会，目前还不清楚，如果每一种文化产生了纯洁的人群。例如，虽然在GM-CSF培养贴壁细胞被描述为巨噬细胞5中 ，从相同的培养非粘附细胞用作树突6,11-13，与推定它们是均匀和任何观察到的变异性是由于发展14,15的不同阶段。此外，研究发现GM-CSF是用于体内 16,17肺泡巨噬细胞发展的一个重要的生长因子，并可以在体外用于生成肺泡状巨噬细胞16,17,18。 除了坚持，从GM-CSF治疗骨髓的文化产生巨噬细胞和DC的过程非常相似的异质性暗示可能GM-CSF骨髓文化中存在。这确实似乎是的情况下为两个论文报告BMDMs在BMDC培养物的非粘附部分的存在。在一个论文中，他们identifi编细胞的CD11c +，CD11b的+ MHCII 中旬 ，MERTK +和CD115 +，它表示最密切相似肺泡巨噬细胞，不得不激活T细胞19的能力下降的基因表达特征的群体。使用MHCII和FL-HA的第二个文件，以便找出一个肺泡巨噬细胞样的人口（的CD11c +，MHCII 中/低 ，FL-HA 高点 ），这是由不成熟的不同（的CD11c +，MHCII 中旬 ，FL-HA 低点 ）和成熟的DC（CD11c的+，MHCII 高 ），这两个表型和功能18。这些论文都表明，GM-CSF BMDC文化是异质的，含有巨噬细胞和DC群表示从BMDC文化解读数据时，应采取照顾。 这个协议描述如何分离骨髓，培养骨髓细胞在GM-CSF，以及识别肺泡巨噬细胞样人口由在骨髓培养未成熟和成熟DC通过流式细胞术采用FL-HA结合和MHCII表达。这个过程是基于Lutz 等人 6的既定的程序，并能产生5 -在一个10毫升培养的第7天10×10 6个非贴壁细胞。培养是可用的从数天7到10和在7这提供了一种简单的方法来生长，并在大量隔离在体外肺泡状巨噬天产生巨噬细胞，未成熟和成熟DC，以及一些前体细胞的异质群体。

<table>
	<tbody>
		<tr>
			<td>Flow Cytometer</td>
			<td>BD&#160;</td>
			<td>LSR-II</td>
			<td></td>
		</tr>
		<tr>
			<td>Automated Inverted Microscope&#160;</td>
			<td>Leica&#160;</td>
			<td>DMI4000 B</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge&#160;</td>
			<td>Thermo Fisher</td>
			<td>ST-40R</td>
			<td></td>
		</tr>
		<tr>
			<td>Biosafety Cabinet</td>
			<td>Nuaire</td>
			<td>NU-425-600</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe 1 ml</td>
			<td>BD</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr>
			<td>26 1/2 Gauge Needle</td>
			<td>BD</td>
			<td>305111</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml Conical Tube&#160;</td>
			<td>Corning</td>
			<td>357070</td>
			<td>*Falcon brand</td>
		</tr>
		<tr>
			<td>Eppendorf tubes (1.5 ml)</td>
			<td>Corning</td>
			<td>MCT-150-C</td>
			<td></td>
		</tr>
		<tr>
			<td>5 ml polystyrene round bottomed tubes</td>
			<td>Corning</td>
			<td>352052</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection Tools</td>
			<td>Fine Science Tools&#160;</td>
			<td>*Various&#160;</td>
			<td>*Dissection scissors, dumont forcep and standard forcep&#160;</td>
		</tr>
		<tr>
			<td>Hemocytometer&#160;</td>
			<td>Richert</td>
			<td>1490</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile 100 x 15 mm Petri Dish</td>
			<td>Corning</td>
			<td>351029</td>
			<td>*Falcon brand</td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Thermo Fisher</td>
			<td>21985-023</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Chloride</td>
			<td>BDH</td>
			<td>BDH0208-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Fisher Bioreagents</td>
			<td>BP1600-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Brefeldin A</td>
			<td>Sigma</td>
			<td>B7651-5MG</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma</td>
			<td>E5134-1KG</td>
			<td>Ethylenediaminetetraacetic acid</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Thermo Fisher</td>
			<td>16000-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#39;s Balanced Salt Solution</td>
			<td>Thermo Fisher</td>
			<td>14175-095&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Thermo Fisher</td>
			<td>15630-080</td>
			<td>4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Sigma</td>
			<td>G8540-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>LPS Ultrapure</td>
			<td>Invivogen</td>
			<td>tlrl-3pelps</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Non-Essential Amino Acids Solution&#160;</td>
			<td>Thermo Fisher</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin 100x</td>
			<td>Thermo Fisher</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Phosphate Monobasic</td>
			<td>BDH</td>
			<td>BDH0268-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>BDH</td>
			<td>BDH9258-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant GM-CSF</td>
			<td>Peprotech</td>
			<td>315-03-A</td>
			<td></td>
		</tr>
		<tr>
			<td>Rooster Comb Sodium Hyaluronate&#160;</td>
			<td>Sigma</td>
			<td>H5388-1G</td>
			<td>*Used to make fluoresceinated hyaluronan</td>
		</tr>
		<tr>
			<td>RPMI-1640&#160;</td>
			<td>Thermo Fisher</td>
			<td>21870-076</td>
			<td>No sodium pyruvate no glutamine. Warm media to 37oC before using.&#160;</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Fisher&#160;</td>
			<td>5271-10&#160;&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Phosphate Dibasic</td>
			<td>Sigma</td>
			<td>50876-1Kg</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Pyruvate</td>
			<td>Sigma</td>
			<td>P5290-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris(hydroxymethyl)aminomethane</td>
			<td>Fisher Bioreagents</td>
			<td>BP152-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue</td>
			<td>Sigma</td>
			<td>T8154</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Fc Receptor (unlabeled), Tissue Culture Supernatant</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Clone: 2.4G2</td>
		</tr>
		<tr>
			<td>Anti-CD11c PeCy7</td>
			<td>eBioscience</td>
			<td>25-0114-82</td>
			<td>Clone: N418</td>
		</tr>
		<tr>
			<td>Anti-Gr-1 efluor450</td>
			<td>eBioscience</td>
			<td>48-5931-82</td>
			<td>Clone: RB6-8C5</td>
		</tr>
		<tr>
			<td>Anti-MHCII APC</td>
			<td>eBioscience</td>
			<td>17-5321-82</td>
			<td>Clone: M5/114.15.2</td>
		</tr>
		<tr>
			<td>Biotinylated Anti-MerTK</td>
			<td>Abcam</td>
			<td>BAF591</td>
			<td>Goat polyclonal IgG</td>
		</tr>
		<tr>
			<td>Streptavidin PE</td>
			<td>eBioscience</td>
			<td>12-4317-87</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium Iodide</td>
			<td>Sigma</td>
			<td>P4170-25MG</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI (4&#39;,6-diamidino-2-phenylindole)</td>
			<td>Sigma</td>
			<td>D9542-5MG</td>
			<td></td>
		</tr>
	</tbody>
</table>

generation and identification of gm-csf derived alveolar-like macrophages and dendritic cells from mouse bone marrow

巨噬细胞和树突状细胞（DC）是在组织和淋巴器官发挥抵御病原体是重要角色发现先天免疫细胞。然而，它们难以在足够数量的分离，以研究它们的详细，因此， 在体外模型已经制定出来。 体外骨髓来源的巨噬细胞和树突状细胞的培养物用于免疫学研究成熟的和有价值的方法。这里，描述了用于培养和使用该细胞因子粒细胞巨噬细胞集落刺激因子（GM-CSF）的原代小鼠骨髓细胞的单培养识别两者的DC和巨噬细胞的方法。此协议是基于首先由Lutz 等人开发的既定程序。在1999年的骨髓来源的DC。培养是异质的，并且MHCII和荧光标记的透明质酸（FL-HA）是用来区分未成熟和成熟DC的巨噬细胞。这些GM-CSF巨噬细胞亲韦迪体外衍生的巨噬细胞可以非常类似于在两个表型和功能肺泡巨噬细胞的一个方便的来源。

总结了该方法的主要步骤的流程图如图1所示。在培养的不同时间的骨髓培养物密度和形态在图2中示出。在第1天，将细胞小和稀疏，用3天有更多的细胞，一些较大和一些已开始附着。第6天有一定的粘附和非粘附部分（ 图2A）。培养可以从第7天收获- 10与存在于第10天。图2B的CD11c +细胞的比例更高示收获前一天7培?...

Watch this Scientific Journal Video about Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow at JoVE.com

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

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. ...

The overall goal is this procedure is to generate and distinguish alveolar-like macrophages from dendritic cells in in vitro murine bone marrow cell cultures supplemented with GM-CSF. This in vitro method can help answer key questions regarding the function of dendritic cells or macrophages. The main advantage of the technique is that it can generate sufficient cell numbers and it can distinguish between dendritic cells and alveolar-like macrophages.The implications of this technique may extend towards the therapy of pulmonary proteinosis as it facilitates the acquisition of alveolar-like macrophages, which can be used to treat this disease. Generally, individuals new to this method will need practice with isolating the cleaned bones and flushing the bone marrow. Visual demonstration of this method is useful as not all techniques used as easily understood by written instruction alone.15 minutes before beginning the dissection, turn on a biological safety cabinet to purge the cabinet air, and to stabilize the air flow, and clean the cabinet surface with 70%ethanol. When the cabinet is ready, place a mouse on top of one to two paper towels in the prone position and spray the animal with 70%ethanol. Then, with the non-dominant hand, lift the skin around the thoracic area and use scissors to make an incision.Grasping both ends of the incision, carefully pull until the two ends of the incision meet at the stomach. Then place the mouse in the supine position and use one hand to pull the lower half of the skin down until the legs are exposed. Holding the foot, cut the Achilles tendons above the ankle joint and the ligaments connecting the muscles to the foot at the lower end of the tibia to loosen the foot from the leg bones.Keeping the leg raised, remove the muscles and ligaments around the knee joint at the lower end of the femur to severe the thigh muscles from the lower leg. Using forceps, gently pull the loosened muscles away from the fibular and femoral surfaces. Then pressing the scissors in the open position at the hip joint, rotate the leg and gently pull on the femur to separate the leg bone from the hip joint while cutting the leg free from the body.Now holding the leg at the hip end with a sterile forceps, use another forceps to remove the foot from the ankle end. Then holding the distal end of the femur with one forceps and the proximal end of the tibia and fibula with another, carefully bend the tibia and fibula in the opposite direction of the knee joint to separate the lower leg from the femur and patella. Use the forceps to remove the remaining ligaments, muscles, and fibula from the lower leg bones and place the cleaned tibia on an inverted Petri dish lid, containing one to two milliliters of HBSS/FCS.Holding the lower end of the femur with one set of forceps and the patella with another, bend the knee and surrounding tissues in the opposite direction to remove the joint. Then remove any of the remaining connective tissues from the femur and place the bone on the inverted Petri dish lid. When all of the bones have been harvested, use forceps to clean any residual tissue still attached to the bones and immerse the bones in 10 milliliters of HBSS/FCS within the base of the Petri dish.Keeping the dish partially shielded with a sterile lid, cut each of the cleaned bones in half with scissors. Then use a one-milliliter syringe, equipped with a 26.5-gauge needle to flush one milliliter of HBSS/FCS from the dish into the end of each bone piece until all of the red marrow has been liberated. Pass any visible clumps of bone marrow through the syringe several times to form a single cell suspension, followed by thorough mixing with a 10 milliliter pipette.Transfer the cells to a collection tube and rinse the Petri dish one time with five milliliters of HBSS/FCS to collect any residual cells. Then pool the wash in the collection tube and place the cells on ice. To set up a bone marrow cell culture, centrifuge the collected cells and aspirate the supernatant with a sterile glass Pasteur pipette attached to vacuum suction.Loosen the red blood cell-rich pellet with tapping. Then add 10 milliliters of RBC lysis buffer and resuspend the cells with vortexing. After a five-minute, room temperature incubation, arrest the lysis with 10 milliliters of HBSS/FCS and spin down the cells, resuspending the pellet in bone marrow cell medium.Count the number of viable cells by Trypan Blue exclusion and transfer the needed cells into a new tube with bone marrow cell medium. Then collect the cells by centrifugation and resuspend the pellet at a four million cell per milliliter dilution in bone marrow cell medium with 20 nanograms per milliliter of GM-CSF. Next, add 9.5 milliliters of bone marrow cell medium supplemented with 20 nanograms per milliliter of GM-CSF to one sterile bacterial Petri dish per culture and add 500 microliters of cells to the center of each dish.Incubate the cells at 37 degrees Celsius and five percent CO2. On day three, add 10 milliliters of fresh bone marrow medium supplemented with 20 nanograms per milliliter of GM-CSF to each culture, taking care to minimize any disturbances to the cells. On day six, carefully transfer 10 milliliters of cell culture supernatant to a sterile 50 milliliter conical tube.Centrifuge the cells and resuspend the pellet in 10 milliliters of fresh bone marrow cell medium, containing 20 nanograms per milliliter of GM-CSF. Then gently vortex the cell suspension and return the cells to the culture dish. On day one, the bone marrow cells are small and sparse but by day three, the cells have increased in number and size, and some have begun to adhere.By day six, there are more cells and both adherent and non-adherent fractions are observed. The culture can be harvested from day seven to 10 and gated by size and live cells with a higher percentage of CD11c-positive cells obtained from day 10 cultures. At day seven, the collected non-adherent fraction is greatly enriched for cells with a dendritic morphology.The CD11c-positive, GR1-negative populations, harvested on day seven and 10, can be divided into three main populations. Macrophages, immature dendritic cells, and mature dendritic cells by MHCII expression and FL-HA binding. Interestingly, MerTK, although considered to be a macrophage marker, exhibits a robust expression on both macrophage and immature dendritic cell populations, harvested on days seven and 10, with a low level of expression observed on mature dendritic cells.While attempting this procedure, it is important to remember to use sterile techniques throughout the bone marrow harvest and cell culture. Following this procedure, the cells also can be sorted to examine cell populations separately, or they can be stimulated with inflammatory agents and subsequently analyzed for their cytokine production by intracellular staining and flow cytometry. After watching this video, you should have a good understanding of how to isolate bone marrow cells and culture them in GM-CSF to generate both dendritic cells and alveolar-like macrophages.

Research

JoVE Journal

Immunology and Infection

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

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

弓形虫囊肿壁形成激活骨髓衍生的巨噬细胞和Bradyzoite条件

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

科研

免疫与感染

A Cell Free Assay System Estimating the Neutralizing Capacity of GM-CSF Antibody using Recombinant Soluble GM-CSF Receptor

一个细胞检测系统估算的GM - CSF抗体的中和能力，采用重组可溶性GM - CSF受体

BACKGROUNDS: Previously, we demonstrated that neutralizing capacity but not the concentration of GM-CSF autoantibody was correlated with the disease ...

Generation and Labeling of Murine Bone Marrow-derived Dendritic Cells with Qdot Nanocrystals for Tracking Studies

小鼠骨髓来源的树突状细胞与跟踪研究的Qdot纳米晶的生成及标签

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone ...

Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

从老鼠肠道树突状细胞和巨噬细胞的分离和表征

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and ...

Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages

使用骨髓源性巨噬细胞巨噬细胞极化的调查

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

Bone Marrow-derived Macrophage Production

骨髓衍生的巨噬细胞产

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

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

荧光激活细胞分选从小鼠骨髓浆树突状细胞的分离纯化

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

从全血人单核细胞来源的树突状细胞的生成

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

Identification and Isolation of Oligopotent and Lineage-committed Myeloid Progenitors from Mouse Bone Marrow

小鼠骨髓 Oligopotent 和世系髓样祖细胞的鉴定与分离

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for ...

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

从小鼠中分离和培养骨髓衍生树突状细胞的经济有效的方案

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