嗜中性粒细胞的分离协议

Biology

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Summary

嗜中性粒细胞之间的第一个细胞到达炎症免疫反应的工地上,和其职能和机制,已被广泛地在体外研究。我们展示一个标准密度梯度分离法,隔离人类使用市售的分离介质从整个血液中性粒细胞。

Cite this Article

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Oh, H., Siano, B., Diamond, S. Neutrophil Isolation Protocol. J. Vis. Exp. (17), e745, doi:10.3791/745 (2008).

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Abstract

中性粒细胞的多形核粒细胞(PMN)在人类之间的第一个细胞到达炎症免疫反应的部位,最丰富的白细胞。由于其关键作用,在炎症,如中性粒细胞功能的运动,细胞因子的产生,的吞噬功能,和肿瘤细胞的作战广泛的研究。为特征的中性粒细胞的特定功能,其他血细胞分离干净,快速,可靠的方法是可取的,尤其是因为在体外研究中性粒细胞是短命的,应在2-4小时收集。在这里,我们展示了一个标准的密度梯度分离法,从全血使用市售分离介质,是一种混合物,钠metrizoate和右旋糖酐500人中性粒细胞隔离。由分层全血通过离心,密度梯度介质,中性粒细胞层的分离,剩余红​​细胞裂解的过程。细胞,然后洗净,计数,并在缓冲区所需浓度的悬浮。正确执行时,此方法已被证明收益率> 95%嗜中性粒细胞> 95%的可行性的样品。

Protocol

嗜中性粒细胞的分离协议

  1. 将所有试剂置于室温。
  2. 收集的5.0毫升离心管中的中性粒细胞隔离媒体。仔细层5.0毫升的血液分离介质,。执行这一步,缓慢而仔细地,并与介质的表面,以避免混合血液和媒体的枪头。
  3. 500 35分钟的离心力离心20-25℃。血液分离出6个不同组别:血浆,单核细胞,隔离媒体,中性粒细胞,更加孤立的媒体,以及红血细胞沉淀(图1)。如果不明确,这些频段的分离过程是不干净,将需要重复。
  4. 小心地取出前三名层(血浆,单核细胞,并进行隔离媒体),用吸管。这些层的处理。
  5. 仔细吸管嗜中性粒细胞和中性粒细胞下方的隔离媒体所有的层。放置到一个干净的离心管的解决方案。
  6. 没有的Ca 2 + / Mg 2 +的HBSS中稀释的中性粒细胞10毫升的解决方案。反转管几次暂停细胞。
  7. 离心机的中性粒细胞在10分钟的350离心力的解决方案。一个红色的沉淀应在试管底部,包含中性粒细胞和残留的红血细胞(红细胞)。用移液器取出上清液小心,颗粒不被打扰。
  8. 裂解残留的红细胞,加入2 ml红细胞裂解液管。悬浮颗粒,旋涡在小瓶设置3-4。避免增加上述4旋涡设置,因为这可能会导致中性粒细胞激活。它可能是必要的,以涡几秒钟,或以“脉冲”涡旋溶解沉淀。
  9. 250 5分钟的离心力离心管。取出上清液用吸管。重复裂解过程中,如果需要的话。
  10. 没有的Ca 2 + / 加500μL的HBSS +每管。同样,旋涡沉淀重悬在设置3-4。没有的Ca 2 + / Mg 2 +的HBSS中,稀释至10 ml 。
  11. 250 5分钟的离心力离心管。吸出上清液并丢弃。
  12. 250μL的HBSS / HSA的解决方案(2%,HSA)重悬沉淀。然后可以计算细胞,并调整到所需浓度。

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Discussion

密度梯度分离法是用来隔离从全血混合使用metrizoate钠和右旋糖酐500人中性粒细胞。这种方法是基于对Boyum(1968年)的单核白细胞分离法修改费兰特和通(1980)中性粒细胞分离。

从捐赠者收集后,全血用EDTA,柠檬酸或肝素抗凝。由于它们是短命的,中性粒细胞应收集2-4小时内使用。由分层全血通过离心,密度梯度介质,中性粒细胞层的分离,剩余红​​细胞裂解的过程。细胞,然后洗净,计算,并重新悬浮到所需浓度。

如果六阶后的第一个离心步骤不同,分离过程是不干净,将需要重复。清洁分离,确保隔离媒体尚未过期或已被污染。离职,也可能不成功,如果献血者在采血前72小时消耗的酒精或药物。

为了防止在分离过程中的中性粒细胞激活,最好使用无钙的HBSS + / Mg2 +的,因为离子总理细胞已被证明。再悬浮的颗粒也应缓慢进行,并应保持在低中档使细胞不被激活的涡设置。

正确执行时,此方法已被证明收益率> 95%嗜中性粒细胞> 95%的可行性的样品。标签与中性粒细胞特异性标志物CD66b和台盼蓝染料排斥细胞纯度和可行性进行评估。

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Acknowledgments

资金从美国国立卫生研究院R01 HL56621。

Materials

Name Type Company Catalog Number Comments
Lymphocyte Poly(R) Reagent Cedarlane Labs PolymorphprepTM can be used as an alternative
Hank’s Balanced Salt Solution without Calcium Chloride Reagent Invitrogen
Human Serum Albumin Reagent ZLB Bioplasma
Red Cell Lysis Buffer Reagent Roche Group
Centrifuge Tool
Vortexer Tool
Pasteur Pipettes and bulb Tool
PolymorphprepTM Reagent Axis-Shield Alternative reagent to Lymphocyte-Poly (R)
Syringe Filter Other EMD Millipore SLGV033RS Millex-GV, 0.22 μm, PVDF, 33 mm, gamma-sterilizable
Hank’s Balance Salt Solution with Calcium Chloride Reagent Invitrogen

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References

  1. Boyum, A. Separation of leucocytes from blood and bone marrow. Scand. J. Clin. Invest. 21, Suppl 97. 77-83 (1968).
  2. England, J. M., Rowan, R. M. The assignment of values to fresh blood used for calibrating automated blood cell counters. Clin. Lab. Haemat. 10, 203-212 (1988).
  3. Garcia, A. Comparison of two leukocyte extraction methods for cytomegalovirus antigenemia assay. J. Clin. Microbiol. 34, 182-184 (1996).
  4. Ferrante, A., Thong, Y. H. Optimal conditions for simultaneous purification of mononuclear and polymorphonuclear leucocytes from human peripheral blood by the Ficoll-Hypaque method. J. Immunol. Methods. 36, 109-109 (1980).

Comments

21 Comments

  1. what is the rational for using whole blood instead of purified white blood cells or enriched neutrophils for NBT test???

    Reply
    Posted by: Anonymous
    September 28, 2008 - 12:37 PM
  2. Why must HSA and not BSA be used? Do you have references for the effect of ETOH and drugs on separation success, and mechanism of action?

    Reply
    Posted by: Anonymous
    October 29, 2008 - 11:21 AM
  3. BSA can activate neutrophils. Brian Siano, on behalf of Hana Oh and Scott Diamond

    Reply
    Posted by: Brian S.
    October 29, 2008 - 11:43 AM
  4. How many HBSS and how many HSA need for HBSS/HSA solution preparation?

    Reply
    Posted by: Anonymous
    January 21, 2009 - 2:50 PM
  5. hi, why do you use HSA? or what is the action of HSA in buffer?  

    Reply
    Posted by: Anonymous
    May 10, 2009 - 3:14 AM
  6. so many things contradict in the movie vs your text

    Reply
    Posted by: Laura K.
    December 4, 2009 - 4:00 PM
  7. From JoVE editor: video and text are complementary parts of each video-article. They are not necessarily 100% the same. There are things that are better described in video than in text, and vice versa. I think in this case the video part is very comprehensive and provides a very clear demonstration on how to do the experiment.

    Reply
    Posted by: Moshe P.
    December 7, 2009 - 3:22 PM
  8. Are there any other reasons why one will not get 6 bands of separation using Polymorphprep? My separation is still not perfect. Repeatign causes the monocyte layer to lower towards the neutrophils. Any suggestions? Donor blood is not affected as mentioned in your video.

    Reply
    Posted by: Anonymous
    June 9, 2010 - 11:28 PM
  9. Any suggestions of modify this protocol so that it will work with NHP blood?

    Reply
    Posted by: Anonymous
    April 14, 2011 - 6:36 PM
  10. Why is ²% HSA added in the last step? What is the advantage of doing that? And what happens is I dont add it?

    Reply
    Posted by: Alex B.
    July 6, 2011 - 3:59 AM
  11. DŒs HBBS for HBBS/HSA preparation contain Ca AND Mg or only Ca?

    Reply
    Posted by: Marija P.
    November 3, 2011 - 3:20 PM
  12. I believe the question above refers to the last step of resuspension of neutrophils. At this point you could use various types of buffer solutions that suite your specific experimental goals. If trying to create a suspension of neutrophils in a physiological saline buffer, both Ca and Mg can be added.

    Reply
    Posted by: Anonymous
    November 3, 2011 - 3:27 PM
  13. I notice that the HBSS contains no phenol red in your neutrophil isolation method. I have noticed that phenol red is avoided (both during isolation and during experiments) in a number of papers implementing neutrophil isolation techniques. Any idea what phenol red dŒs to neutrophils? I have used a variation on the isolation technique (generously shared by Drs. Oh, Siano and Diamond) and ended by suspending my isolated neutrophils in RPMI with phenol red. Although phenol red might not be the (only) culprit, I am not seeing any neutrophil killing activity (ADCC assay).
    I plan to attempt the method provided in this video later this week. Any ideas about phenol red?

    Reply
    Posted by: Anonymous
    March 19, 2012 - 9:58 AM
  14. We avoid pH indicator dyes because they often cause autofluorescence and lead to high background in fluorescent assays. We don't have any direct measurements of fluorescent dyes on neutrophil function.

    Reply
    Posted by: Anonymous
    March 19, 2012 - 10:10 AM
  15. I have seen that during neutrophil isolation from my blood there is no layer of neutrophils while there is a thick band of ruptured RBC at that position,every time.Can anyone suggest me,what could be the reason for that?

    Reply
    Posted by: Anonymous
    April 3, 2012 - 4:25 AM
  16. I'm getting the same result every time. Did you ever find out what's causing a thick band of RBC at the position of neutrophils bands?

    Reply
    Posted by: Zahra M.
    February 17, 2014 - 12:23 PM
  17. I have been in face of the same problem that part of RBC can not sediment to the bottom band. This almost happpens when centrifuging at 500RCF but hardly happen at 450 RCF. So I guess 500 RCF is inappropriate.

    Reply
    Posted by: Xu L.
    May 25, 2017 - 3:50 AM
  18. Is this protocol applicable to Dog and Rat Neutrophil isolation? If so, do I still use HSA?

    Reply
    Posted by: Vishnu U.
    June 27, 2012 - 3:04 PM
  19. I have a similar question. I am interested in using this protocol to isolate neutrophils from mouse whole blood. Did you ever find out if this protocol works for rat neutrophil isolation?

    Reply
    Posted by: Hanna M.
    January 29, 2014 - 1:59 PM
  20. Hi. It is said in the article that the neutrophil yield would be 2x10^6 per ml. Does that mean 2x10^6 per ml of whole blood used? So using 5 ml of blood (as used in the article) would yield 10 million neutrophils?
    I got an extremely low count for my neutrophils. about 2 million from 5ml blood. Can you please help me troubleshoot?
    Also, why did you mention 10-15ml blood (yielding x10^8 cells) in the beginning? Is it that you carry out the isolation from 10-15 ml blood in 2 or 3 parts of 5 ml each? Please help.
    Thanks.

    Reply
    Posted by: Sumana B.
    September 25, 2014 - 3:17 PM
  21. I a following this protocol, but running into clumping of the cells. Is there any way to prevent this from happening?

    Reply
    Posted by: Anonymous
    August 9, 2017 - 9:52 PM

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