分离和造血干细胞移植(造血干细胞)

Biology

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Summary

Cite this Article

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Lo Celso, C., Scadden, D. Isolation and Transplantation of Hematopoietic Stem Cells (HSCs). J. Vis. Exp. (2), e157, doi:10.3791/157 (2007).

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Abstract

Protocol

共有骨髓的准备

  1. 四B6小鼠牺牲和胫骨,股骨,髋部和脊柱解剖获得。
  2. 在解剖,四肢和骨骼都保存在PBS 2%FCS的热灭活(可选2MM的EDA - 夹层介质)。
  3. 干净的骨头被压碎用迫击炮和杵在夹层介质。做一个有效的方法是粉碎一次每只小鼠的胫骨,股骨和髋部,然后2刺。
  4. 从每个鼠标中获得细胞的混合物被单独存放,并通过50毫升猎鹰管成40微米过滤器过滤。在这种方式中,每管含有细胞的混合物,获得了所有的鼠标,从2棘获得的混合物的一半腿骨。在这个阶段,过滤器是用于单独的骨骼碎片。
  5. 它通常是最好粉碎腿骨两次获得白(无骨髓)碎骨,脊柱片段2或3倍。
  6. 填充管他们平衡,旋转5分钟1200RPM。
  7. 吸上清,松开拖管的管架(每个离心后,以尽量减少聚集形成和细胞的损失,这一步很重要)沉淀。重悬在每管1毫升PBS 2%FCS(没有EDTA从现在开始,)如果你正在做血统的枯竭,或在任何音量,否则您方便。

天堂枯竭

  1. 此过程可以消除高度分化的细胞,并获得一个非常庞杂的人口细胞,干细胞和祖细胞丰富。如果你是排序的HSC,这一步大幅降低的细胞通过细胞分选去的数量,从而减少排序的时间,并允许排序的HSC较高的小鼠,让你可以得到一个较高的数字的HSC在一个单一的排序。
  2. 如果你要排序后,取出的细胞混合排序控件(未染色和SCA1,CD48,c - Kit的Flk2单一的颜色控制)(我通常采取从每个管位)50μL。
  3. 新增林鸡尾酒30ml/tube。天堂鸡尾酒含有多种抗体,不幸变化不大,从实验室到实验室。在Scadden实验室,我们使用biotinilated抗体对GR1,Ter119,CD4 +,CD8 +,CD3 +,B220。他们在染色的最终稀释度为1:700。
  4. 涡迅速混合,孵育15分钟,在4 ° C。
  5. 在孵化期间,准备上的磁铁和下15毫升洗脱列管的列。德加一些PBS使用steriflip过滤器(你应该看到迈向由于负压吸引的PBS表面的小气泡),和平衡3毫升脱气PBS每一列列。
  6. 柱流量约1ml/5min,所以准备使用的列,它大约需要15分钟。
  7. 在孵化结束,加PBS洗涤2%FCS的填补管和旋转5分钟,在1200RPM。
  8. 吸出上清液,松开颗粒和悬浮在1毫升/脱气PBS管。
  9. 如果你是排序后,取出天堂单一颜色的染色CTRL总骨髓(15毫升,每管位)。
  10. 添加30ml/tube链亲和素珠,涡迅速混合,孵育15分钟,在4 ° C。
  11. 在孵化结束,加2毫升/管脱气PBS。把下列新的收集管,并适用于每一个暂停一列,通过40微米的过滤器进行过滤。
  12. 每管加入另3毫升脱气PBS洗。一旦列是空的,再次申请使用相同的40微米过滤器。
  13. 洗脱液包含枯竭的谱系细胞的混合物,异构的人口为干细胞和祖细胞丰富。游泳池的内容分为2管,4管。在1500RPM旋转5分钟。
  14. 吸上清。颗粒应为白色或白色为主。如果你是排序后松开颗粒1毫升总PBS 2%FCS一起重悬,否则悬浮在任何音量方便您。

LKS或的长远的HSC排序

  1. 取出血统耗尽单一颜色的染色CTRL(15毫升)。这将允许您评估您的血统枯竭的效率。
  2. 15毫升管染色细胞混合排序。

新增抗体:

SCA PE - Cy5.5 1:100 10毫升
包APC 1:100 10毫升
SA的PE - Cy7 1:500 2毫升

要排序长期的HSC,还可以添加:

CD48 FITC 1:1000 1毫升
Flk2体育 1:200 5毫升

此外,准备单染色控制使用的总预留保持解剖和骨髓血统染色后的骨髓。这些染色法,流式细胞仪管可以直接到。

确保在黑暗中!

  1. 涡迅速混合,并在4 ° C时20至30分钟,再搅拌中途孵化。
  2. 洗去多余的抗体,填补了PBS 2%FCS和自旋为5分钟1500转管。
  3. 去除上清液,重新悬浮颗粒和移动新流式细胞仪管过滤器盖。它可能需要一段时间来获得的细胞经过这些过滤器,重要的是申请多一些PBS事后冲洗过滤器,最大限度地减少细胞损失。这一步是必要的,以避免堵塞的分拣机,这是最可怕的噩梦的每一个细胞分选仪用户。
  4. 当交给你的细胞,细胞分选设施,确保还准备与PBS 10%您好FCS的细胞,将收集管。
  5. 如果你是李嘉诚细胞排序,您将获得一个包含长期和短期的复育HSC和多向祖细胞的异质人口。从4只小鼠的平均产量是30万李嘉诚。
  6. 如果你也用CD48和Flk2抗体,可以单独每一个长期的HSC,短期HSC和MPP人口。 LT - HSC是最难得的人口。平均产量约5万至6万4小鼠的细胞。

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Comments

9 Comments

  1. Hi, Thankyou for the wondeful post. I was working with endothelial progenitor cells and wanted to know how many of the bone marrow cells are Sca-1 positive. Also is it possible that we can extract the BMC and then store them at -²0 and stain them for FACS the next day? Any help will be greatly appreciated. Thanks 

    Reply
    Posted by: Anonymous
    March 31, 2008 - 1:45 PM
  2. You can sort on the following day, but you should keep the cells at 4C, better in an ice bucket in the fridge. Many cells die upon freezing

    Reply
    Posted by: Anonymous
    April 17, 2009 - 10:25 AM
  3. Can you please tell me where you purchased your streptavidin beads? Thank you.

    Reply
    Posted by: Anonymous
    April 2, 2008 - 12:33 PM
  4. miltenyi

    Reply
    Posted by: Anonymous
    April 17, 2009 - 10:27 AM
  5. How do you isolate muscle tissue from spin bone? And if without specific step to isolate muscle tissue, would the results of FACS be different? BTW. How do you deleption RBC form those bone marrows? Thanks.

    Reply
    Posted by: Anonymous
    December 23, 2008 - 4:20 AM
  6. you need to use a scalpel and much patience. LEaving muchmuscle around the spine makes it harder to crush and will reduce your yield.   RBC are depleted while lineage depleting. The post-column pellet is white. I do no recommend a separate RBC lysis because there is the risk it will damage other cells also.Moreover, it is hard to perform it consistently.

    Reply
    Posted by: Anonymous
    April 17, 2009 - 10:29 AM
  7. How many HSCs do you inject into the irradiated recipients?

    Reply
    Posted by: Wilma J.
    August 20, 2010 - 11:39 AM
  8. Hi,
    My name is shahid chaudhary and I am working on EAE mice. The only problem i am facing with the experiment is the intravenous injection of genetically modifed HSC into the tail of the mice. I have used lamps but its not helping me out and because of this I am feeling very underestimated while doing the expperiment. Can anyone suggest me some good tips regarding the intravenous tail vein injection. That will be very much grateful.

    Reply
    Posted by: Shahid C.
    December 5, 2012 - 10:30 AM
  9. Hi,
    My name is shahid chaudhary and I am working on EAE mice. The only problem i am facing with the experiment is the intravenous injection of genetically modifed HSC into the tail of the mice. I have used lamps but its not helping me out and because of this I am feeling very underestimated while doing the expperiment. Can anyone suggest me some good tips regarding the intravenous tail vein injection. That will be very much grateful.

    Reply
    Posted by: Shahid C.
    December 5, 2012 - 10:30 AM

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