Neutrophil Isolation from the Intestines
从肠道中分离嗜中性白细胞   

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参见作者原研究论文

本实验方案简略版
Nature Medicine
Jun 2014

 

Abstract

This protocol provides the possibility to isolate leukocytes including neutrophils out of intestinal tissues to use the received cells in further experiments of interest.

Materials and Reagents

  1. HEPES (Serva, catalog number: 25245 )
  2. EDTA (Merck KGaA, catalog number: 108418 )
  3. Dispase in HBSS (BD Bioscience, catalog number: 354235 )
  4. Collagenase D (Roche Diagnostics, catalog number: 11088866001 )
  5. DNAse I (Sigma-Aldrich, catalog number: DN25 )
  6. 4% (v/v) FCS (PAN Biotech, catalog number: P30-3302 )
  7. Ice-cold PBS (Gibco, catalog number: 14190-094 )
  8. HBSS (Gibco, catalog number: 14175-053 )
  9. Percoll (GE Healthcare, catalog number: 17-0891-01 )
  10. 10x PBS (Ambion, catalog number: AM9624 )
  11. RPMI (Gibco, catalog number: 21875-034)
  12. Cell-dissociation buffer (CD buffer) (see Recipes)
  13. Digestion solution (see Recipes)
  14. 40% percoll (see Recipes)
  15. 80% percoll (see Recipes)

Equipment

  1. Incubator (New Brunswick Scientific, incubator shaker G25)
  2. 15 ml tube (Greiner Bio-One, catalog number: 188161 )
  3. Centrifuge (Thermo Scientific, model: Multifuge X1R )

Procedure

  1. After removal, the small intestine was freed from surrounding connective tissue and cut open longitudinally in order to wash out feces by ice-cold PBS.
  2. To focus on lamina propria leukocytes, the intraepithelial leukocytes and the epithelial layer were removed by use of cell-dissociation buffer (CD buffer).
    Note: Several factors including tissue type and pH influence the effectiveness of cell dissociation procedures.Here, we used EDTA as a chelator and HEPES as a buffer component for gentle cell dissociation.
  3. The intestines were incubated with 10 ml CD buffer for 10 min at 37 °C and 100 rpm.
  4. The samples were intensively vortexed, the intestine was transferred into 10 ml fresh CD buffer and the incubation for 10 min at 37 °C and 100 rpm was repeated.
  5. The samples were intensively vortexed, the intestines were cut into small pieces with a scalpel and 5 ml of digestion solution was added.
  6. The samples were incubated for 20 min at 37 °C and 100 rpm, vortexed, let the undigested tissue collect at the ground by sedimentation and carefully transferred the liquid phase in an ice-cold collection tube.
  7. Another 5 ml of digestion solution was added to undigested intestinal tissue, the incubation for 20 min at 37 °C and 100 rpm and the liquid phase collection cycle were repeated another two times.
  8. Intestinal leukocytes containing neutrophils were separated from stromal cells (which were dislodged from the lamina propria along with the leukocytes) with a percoll gradient.
  9. The digested intestines were centrifuged at 4 °C (10 min, 450 x g), the supernatant was discarded carefully and the pellet was resuspended in 5 ml 40% percoll.
  10. This suspension was carefully loaded on 5 ml 80% percoll in a 15 ml tube, creating a 40%/ 80% gradient with a sharp border.
  11. The gradient was centrifuged at 4 °C (20 min, 700 x g) with a gentle acceleration and without using the brakes of the centrifuge.
  12. The interphase (approximately 5 ml), containing lamina propria leukocytes, was harvested. Out of a small intestine of one healthy mouse up to 10 million leukocytes can be isolated.
  13. Cells should be washed with PBS to clean out the residue percoll.
  14. The purity of the cells can be checked by a flow cytometric staining and analysis.

Representative data

Several millions of leukocytes can be isolated out of the intestinal tract of a healthy mouse.

Notes

Fast working and cooling of the collected cells on ice optimizes the number of isolated leukocytes. Cells should be washed with PBS before proceeding to the experiments of interest to wash out the percoll.

Recipes

  1. Cell-dissociation buffer (CD buffer)
    10 mmol/L HEPES
    6.4 mmol/L EDTA
    Solve in HBSS
  2. Digestion solution
    250 U Dispase in HBSS
    2.5 mg Collagenase D
    2.5 mg DNAse I
    4% (v/v) FCS
  3. 40% percoll
    40% (v/v) percoll and 10% (v/v) 10x PBS in RPMI
  4. 80% percoll
    80% (v/v) percoll and 10% (v/v) 10x PBS in RPMI

Acknowledgments

This work was supported by the DFG (ZE872/1-2 individual grant to RZ).

References

  1. Schwab, L., Goroncy, L., Palaniyandi, S., Gautam, S., Triantafyllopoulou, A., Mocsai, A., Reichardt, W., Karlsson, F. J., Radhakrishnan, S. V., Hanke, K., Schmitt-Graeff, A., Freudenberg, M., von Loewenich, F. D., Wolf, P., Leonhardt, F., Baxan, N., Pfeifer, D., Schmah, O., Schonle, A., Martin, S. F., Mertelsmann, R., Duyster, J., Finke, J., Prinz, M., Henneke, P., Hacker, H., Hildebrandt, G. C., Hacker, G. and Zeiser, R. (2014). Neutrophil granulocytes recruited upon translocation of intestinal bacteria enhance graft-versus-host disease via tissue damage. Nat Med 20(6): 648-654.

简介

该方案提供了将白细胞包括嗜中性粒细胞从肠组织中分离的可能性,以在进一步的感兴趣的实验中使用接收的细胞。

材料和试剂

  1. HEPES(Serva,目录号:25245)
  2. EDTA(Merck KGaA,目录号:108418)
  3. Dispase在HBSS(BD Bioscience,目录号:354235)中
  4. 胶原酶D(Roche Diagnostics,目录号:11088866001)
  5. DNAse I(Sigma-Aldrich,目录号:DN25)
  6. 4%(v/v)FCS(PAN Biotech,目录号:P30-3302)
  7. 冰冷PBS(Gibco,目录号:14190-094)
  8. HBSS(Gibco,目录号:14175-053)
  9. Percoll(GE Healthcare,目录号:17-0891-01)
  10. 10x PBS(Ambion,目录号:AM9624)
  11. RPMI(Gibco,目录号:21875-034)
  12. 细胞解离缓冲液(CD缓冲液)(参见配方)
  13. 消解解决方案(参见配方)
  14. 40%percoll(见配方)
  15. 80%percoll(见食谱)

设备

  1. 培养箱(New Brunswick Scientific,恒温摇床G25)
  2. 15ml管(Greiner Bio-One,目录号:188161)
  3. 离心机(Thermo Scientific,型号:Multifuge X1R)

程序

  1. 移除后,小肠从周围的结缔组织释放,并纵向切开以便通过冰冷的PBS洗出粪便。
  2. 为了关注固有层白细胞,使用细胞解离缓冲液(CD缓冲液)除去上皮内白细胞和上皮层。
    注意:包括组织类型和pH的几个因素影响细胞解离过程的有效性。因此,我们使用EDTA作为螯合剂,HEPES作为缓冲细胞解离的缓冲成分。
  3. 将肠与10ml CD缓冲液在37℃和100rpm下温育10分钟
  4. 将样品强烈涡旋,将肠转移到10ml新鲜CD缓冲液中,并在37℃和100rpm下重复孵育10分钟。
  5. 将样品剧烈涡旋,用解剖刀将肠切成小块,并加入5ml消化溶液。
  6. 将样品在37℃和100rpm下孵育20分钟,涡旋,使未消化的组织通过沉降在地面上收集,并在冰冷的收集管中小心转移液相。
  7. 将另外5ml的消化溶液加入到未消化的肠组织中,在37℃和100rpm下温育20分钟,并将液相收集循环重复两次。
  8. 含有中性白细胞的肠白细胞与基质细胞(其与白细胞一起从固有层中移出)与percoll梯度分离。
  9. 将消化的肠在4℃下离心(10分钟,450xg),小心弃去上清液,将沉淀重悬于5ml 40%percoll中。
  10. 将该悬浮液小心地装载在15ml管中的5ml 80%percoll上,产生具有尖锐边界的40%/80%梯度。
  11. 将梯度在4℃(20分钟,700×g)下以轻微的加速度离心,不使用离心机的制动器。
  12. 收获含有固有层白细胞的中间相(约5ml)。 在一个健康小鼠的小肠中,可以分离出高达1000万个白细胞
  13. 细胞应用PBS洗涤以清除残留物percoll
  14. 可以通过流式细胞染色和分析检查细胞的纯度

代表数据

数百万白细胞可以从健康小鼠的肠道中分离出来。

笔记

在冰上收集的细胞的快速工作和冷却优化了分离的白细胞的数量。 在进行目的实验以洗出Percoll之前,应用PBS洗涤细胞。

食谱

  1. 细胞解离缓冲液(CD缓冲液)
    10 mmol/L HEPES
    6.4 mmol/L EDTA
    在HBSS中解决
  2. 消化解决方案
    250 H在HBSS中的释放
    2.5mg胶原酶D
    2.5mg DNAse I
    4%(v/v)FCS
  3. 40%percoll
    40%(v/v)percoll和10%(v/v)10x PBS的RPMI中
  4. 80%percoll
    80%(v/v)percoll和10%(v/v)10x PBS的RPMI中

致谢

这项工作是由DFG(ZE872/1-2个人授权RZ)支持。

参考文献

  1. Schwab,L.,Goroncy,L.,Palaniyandi,S.,Gautam,S.,Triantafyllopoulou,A.,Mocsai,A.,Reichardt,W.,Karlsson,FJ,Radhakrishnan,SV,Hanke, Graeff,A.,Freudenberg,M.,von Loewenich,FD,Wolf,P.,Leonhardt,F.,Baxan,N.,Pfeifer,D.,Schmah,O.,Schonle,A.,Martin,SF,Mertelsmann R.,Duyster,J.,Finke,J.,Prinz,M.,Henneke,P.,Hacker,H.,Hildebrandt,GC,Hacker,G.and Zeiser,R。(2014)。 在肠细菌移位时募集的中性粒细胞通过组织损伤增强移植物抗宿主病。 a> Nat Med 20(6):648-654。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Goroncy, L. and Zeiser, R. (2015). Neutrophil Isolation from the Intestines. Bio-protocol 5(6): e1420. DOI: 10.21769/BioProtoc.1420.
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