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
Summary
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.
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Flow Cytometer | BD | LSR-II | |
| Automated Inverted Microscope | Leica | DMI4000 B | |
| Centrifuge | Thermo Fisher | ST-40R | |
| Biosafety Cabinet | Nuaire | NU-425-600 | |
| Syringe 1 ml | BD | 309659 | |
| 26 1/2 Gauge Needle | BD | 305111 | |
| 50 ml Conical Tube | Corning | 357070 | *Falcon brand |
| Eppendorf tubes (1.5 ml) | Corning | MCT-150-C | |
| 5 ml polystyrene round bottomed tubes | Corning | 352052 | |
| Dissection Tools | Fine Science Tools | *Various | *Dissection scissors, dumont forcep and standard forcep |
| Hemocytometer | Richert | 1490 | |
| Sterile 100 x 15 mm Petri Dish | Corning | 351029 | *Falcon brand |
| 2-Mercaptoethanol | Thermo Fisher | 21985-023 | |
| Ammonium Chloride | BDH | BDH0208-500G | |
| Bovine Serum Albumin | Fisher Bioreagents | BP1600-1 | |
| Brefeldin A | Sigma | B7651-5MG | |
| EDTA | Sigma | E5134-1KG | Ethylenediaminetetraacetic acid |
| Fetal Bovine Serum | Thermo Fisher | 16000-044 | |
| Hank's Balanced Salt Solution | Thermo Fisher | 14175-095 | |
| HEPES | Thermo Fisher | 15630-080 | 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid |
| L-Glutamine | Sigma | G8540-100G | |
| LPS Ultrapure | Invivogen | tlrl-3pelps | |
| MEM Non-Essential Amino Acids Solution | Thermo Fisher | 11140-050 | |
| Penicillin/Streptomycin 100x | Thermo Fisher | 15140-122 | |
| Potassium Phosphate Monobasic | BDH | BDH0268-500G | |
| Potassium Chloride | BDH | BDH9258-500G | |
| Recombinant GM-CSF | Peprotech | 315-03-A | |
| Rooster Comb Sodium Hyaluronate | Sigma | H5388-1G | *Used to make fluoresceinated hyaluronan |
| RPMI-1640 | Thermo Fisher | 21870-076 | No sodium pyruvate no glutamine. Warm media to 37oC before using. |
| Sodium Chloride | Fisher | 5271-10 | |
| Sodium Phosphate Dibasic | Sigma | 50876-1Kg | |
| Sodium Pyruvate | Sigma | P5290-100G | |
| Tris(hydroxymethyl)aminomethane | Fisher Bioreagents | BP152-5 | |
| Trypan Blue | Sigma | T8154 | |
| Anti-Fc Receptor (unlabeled), Tissue Culture Supernatant | N/A | N/A | Clone: 2.4G2 |
| Anti-CD11c PeCy7 | eBioscience | 25-0114-82 | Clone: N418 |
| Anti-Gr-1 efluor450 | eBioscience | 48-5931-82 | Clone: RB6-8C5 |
| Anti-MHCII APC | eBioscience | 17-5321-82 | Clone: M5/114.15.2 |
| Biotinylated Anti-MerTK | Abcam | BAF591 | Goat polyclonal IgG |
| Streptavidin PE | eBioscience | 12-4317-87 | |
| Propidium Iodide | Sigma | P4170-25MG | |
| DAPI (4',6-diamidino-2-phenylindole) | Sigma | D9542-5MG |
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