文化髓系树突状细胞从骨髓前体

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Summary

这个视频演示的区分从小鼠骨髓髓系树突状细胞的过程。隔离表明小鼠的胫骨和股骨,加工和骨髓。图片展示前和分化后的细胞形态和数字描绘细胞表型和IL - 12的生产以下成熟使用的CpG所示。

Cite this Article

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Boudreau, J., Koshy, S., Cummings, D., Wan, Y. Culture of myeloid dendritic cells from bone marrow precursors. J. Vis. Exp. (17), e769, doi:10.3791/769 (2008).

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Abstract

髓系树突状细胞(DCS)是经常被用来研究先天免疫和适应性免疫机制和感染的早期反应之间的相互作用。因为这些都是最有力的抗原提呈细胞,区议会正越来越多地使用作为疫苗载体的研究抗原特异性免疫应答的诱导。在这段视频中,我们展示了收获来自捐助者鼠标的胫骨和股骨,处理骨髓,在体外鉴别区议会的程序。区议会的变化刺激未成熟树突状细胞后的属性是强有力的吞噬细胞,而成熟DC抗原提呈和+和CD8 + T细胞与CD4的相互作用。这种功能活动的变化对应与上调细胞表面标志和细胞因子的产生。很多代理商可以使用成熟的区议会,包括细胞因子和Toll样受体配体。在这段视频中,我们展示了两个共刺激分子,CD86和CD40分子,细胞因子,IL - 12的表达,流式细胞仪比较,与中央人民政府或模拟处理过夜刺激。分化后,区议会可以进一步操纵使用或作为疫苗载体在体外产生抗原特异性免疫应答脉冲肽或蛋白质,或转用重组病毒载体。

Protocol

此协议已经适应卢茨等。

收获和加工骨髓

  1. 胫骨与股骨的隔离 :安乐死捐助鼠标,喷以70%乙醇的腿。牢牢抓住脚踝,钝钳和开始剪去的皮肤和基础肌肉暴露胫骨。为了避免损坏骨,缓慢切割和并行的胫骨,而使膝关节完好。

    清洁股骨,抓钝钳固定膝关节。清洁干净,一双锋利的剪刀,弯钳的肌肉。继续向上,直到髋关节暴露和剪刀,可置于股骨的头部和髋关节之间。取出的骨头,股骨和髋关节和地方之间的切割到磷酸缓冲液(PBS)在冰上。
  2. 去除骨髓:清洁,其余用剪刀和弯钳骨骼肌肉。切断每个骨的骨骺和定位的中心腔。使用10ml注射器,用PBS加载和25G0.5“针,骨髓冲入非组织涂层的培养皿。
  3. 骨髓加工:从1ml注射器用橡胶活塞结束分解成单细胞悬液骨髓(使用向上和向下,而不是刮的议案)。收集在一个圆锥形的猎鹰管的骨髓,用PBS冲洗残留的骨髓。

树突状细胞的培养

  1. 一个血球悬浮细胞(图1)直流媒体和计数直流前兆。你会看到大小不等的细胞;的DC前体是最大的和最聪明的。差异数只有这些细胞。你应该从两个股骨和胫骨的两个野生型C57BL / 6小鼠(6-8周龄),预计25-40 × 10 6的总DC前体之间。



    图1

  2. 在培养细胞密度2 × 10 5 DC前体/直流媒体毫升40毫微克/毫升的重组小鼠GM - CSF的补充。在非组织涂层的聚苯乙烯培养皿板的板细胞。

新增媒体(3天)

要刷新的媒体,辅以40毫微克/毫升GM - CSF的新媒体总量的一半。

更换三分之一的媒体(6天)和成熟

要刷新的媒体,小心地取出媒体总量的三分之一,并取代与文化的第6天与40 ng / mL的GM - CSF的补充新鲜直流媒体音量。

如果需要,可以刺激树突状细胞,用细胞因子或Toll样受体配体的成熟。在录像中,区议会是成熟的5毫微克/毫升的CpG一夜之间刺激。

树突状细胞的收获

DC文化是完整的(图2)。细胞就会被悬挂和松散坚持板块。坚持细胞与组织文化刮板刮碟,用PBS漂洗可去除。在为期一周的文化和分化期细胞总数将增加5-8倍,因此,预计收获1-1.6 × 10 6细胞/ ml。

图2

图2

试剂

  1. 直流媒体
    1. RPMI - 1640的媒体,辅以:
    2. 10%胎牛血清
    3. 100 IU / mL青霉素
    4. 100μg/ mL链霉素
    5. 1%的L -谷氨酰胺
    6. 0.1%2 - 巯基乙醇
    7. 1X非必需氨基酸
    8. 1X丙酮酸钠
  2. 重组小鼠GM - CSF的
    1. rmGM - CSF(Peprotech公司,新泽西州,目录没有。315-03)

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Discussion

区议会是有用的先天免疫和适应性免疫的相互作用的研究,可受聘作为疫苗载体。在这个视频中,我们已经证明的步骤来隔离骨髓和髓系树突状细胞在体外分化。文化时期之后,这些细胞可以被可视化微观为贴壁细胞,它往往具有树突,以及非贴壁的圆形细胞。区议会可以进一步操纵抗原提呈抗原脉冲刺激的细胞因子的生产和使用细胞因子和/或Toll样受体配体(检讨,看到吉尔博亚,2008(2))共刺激分子的上调。

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Acknowledgements

巴顿是由自然科学和工程研究理事会(NSERC)的奖学金支持。为支持这一项目由加拿大卫生研究院提供,批准“公约”,67066。

Materials

Name Type Company Catalog Number Comments
recombinant murine GM-CSF Reagent Peptrotech 315-03

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References

  1. Lutz, M. B., Kukutsch, N., Ogilvie, A. L., Rossner, S., Koch, F., Romani, N., Schuler, G. An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow. J. Immunol. Methods. 223, 77-92 (1999).
  2. Gilboa, E. DC-based cancer vaccines. J. Clin. Invest. 117, 1195-1203 (2007).

Comments

14 Comments

  1. Adherent cells are macrophages not DCs. They are >95% F4/80 positive macrophages.

    Reply
    Posted by: Anonymous
    December 12, 2008 - 1:21 PM
  2. I confirm that!

    Reply
    Posted by: Imane A.
    October 10, 2012 - 6:12 AM
  3. The expression level of Class II molecules is strong? Have you tested the level of this marker in your cells?

    Reply
    Posted by: Anonymous
    January 30, 2009 - 10:27 AM
  4. These cells are generally about ²5% MHC II+ and expression is increased >65% after stimulation (ie with LPS)

    Reply
    Posted by: Jeanette B.
    May 6, 2009 - 6:39 PM
  5. How long can dendritic cells be cultured for before use in an experiment? How often should media be changed if you are not using them immediately?

    Reply
    Posted by: Sarah L.
    May 5, 2009 - 3:48 PM
  6. I have never tried to culture them longer than 10 days, but they are healthy at that point. Maybe someone else has? If you do culture them longer, they should be split 1/² or 1/3 (keep the media that they're in and top up with fresh media) and more frequently if the media begins to yellow. They grow quickly at this stage!    

    Reply
    Posted by: Jeanette B.
    May 6, 2009 - 6:43 PM
  7. Do you know if splitting the cells will activate them? I am afraid I'll lose a lot of cells... (not sure if I should expect all of them to re-attach or not...)

    Reply
    Posted by: Anonymous
    May 12, 2010 - 3:43 PM
  8. 14 days.

    Reply
    Posted by: Imane A.
    October 10, 2012 - 6:14 AM
  9. when i culture the cells i often see a few small (maybe 1 or ²mm) of cells. It is visible to the eye and just looks like a clump of cells under the microscope. I use GM-CSF to mature the cells but nothing to stimulate them as i have to do that as part of my experiment at a later date. I was just wondering is it normal for the cells to clump like that if they are not activated?

    Reply
    Posted by: Anonymous
    June 5, 2009 - 9:48 AM
  10. It is normal to have these small clusters early in the culture period. They should be very rare by day 7 of culture, even after stimulation.

    Reply
    Posted by: Jeanette B.
    June 6, 2009 - 9:30 AM
  11. Hi, just like to ask...Should I just collect the non-adherent cells after GM-CSF differentiation for subsequent culturing if the adherent cells are macrophages?
    I intend to follow the protocol of Lutz and the whole generation of DCs will take a period of 1² days. I hope the cells can survive for a long period.

    Reply
    Posted by: Anonymous
    June 18, 2009 - 5:30 AM
  12. why do you not use IL-4 + rmGM-CSF?, and why do you use 40 ng/mL of GM-CSF, if Luzt use ²0ng/ml? Where do you mature the DC, in the same plate?

    Reply
    Posted by: paula c.
    August 15, 2009 - 10:38 AM
  13. I work with Sprague Dawley Rats and my experience is that when I use the combination of GMCSF and IL4 the capacity of the cells to stimulate T cell activation/diferrentiation decrease.....in comparisson withthe ones culture just with GMCSF

    Reply
    Posted by: Anonymous
    August 21, 2009 - 9:10 AM
  14. When you replace/change the media...Are you lossed a lot of cells????

    Reply
    Posted by: Anonymous
    August 21, 2009 - 9:13 AM

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