Improve Research Reproducibility A Bio-protocol resource
  • search
  • 提交稿件
  • 订阅
  • CN change language
Peer-reviewed

Isolation of Cells from Human Intestinal Tissue

从人小肠组织分离细胞

Heli Uronen-Hansson Heli Uronen-Hansson
EP Emma Persson
PN Petra Nilsson
WA William Agace

发布: 2014年04月05日第4卷第7期 DOI: 10.21769/BioProtoc.1092 浏览次数: 17276

评审: Ivan ZanoniSavita NairAnonymous reviewer(s)

download pdf 下载 PDF 下载 PDF
ask Q&A
引用
收藏
Cited by

参见作者原研究论文

The authors used this protocol in:

Cover of Immunity, featuring study using the protocol.

May 2013

Presubmission Inquiry

实验方案合集

专注于特定技术或应用的、详细的、经过同行评审的实验方案合集

Cancer Research
Immunology
Developmental Biology
Microbiology
Neuroscience
Cell Imaging - A Special Collection for Cell Bio 2023
查看更多 more

相关实验方案

小鼠原代周细胞的分离和培养

小鼠原代周细胞的分离和培养

Tamara McErlain [...] Meera Murgai

2025年04月20日 1682 阅读

小鼠脉络膜内皮细胞的原代培养方法

小鼠脉络膜内皮细胞的原代培养方法

Qiuhua Yang [...] Yuqing Huo

2025年06月20日 843 阅读

基于磁激活细胞分选（MACS）的小鼠肾足细胞分离方法

基于磁激活细胞分选（MACS）的小鼠肾足细胞分离方法

Jeffrey W. Pippin [...] Stuart J. Shankland

2025年07月05日 1176 阅读

Abstract

The intestinal lamina propria contains a dense network of T cells, dendritic cells (DCs) and macrophages, which play an important role in local innate and adaptive immune responses. We have recently identified distinct subsets of DCs (Persson et al., 2013) and macrophages (Bain et al., 2013) in the human intestine. In addition, we have studied T cells in healthy and diseased intestine. Here, we describe two methods for isolating these cell populations: 1) enzymatic treatment and 2) migration based isolation. The enzymatic method can be used to isolate T cells, DC and macrophages, whereas the migration based ‘walk-out’ protocol is suitable for DC isolation, as these cells migrate out from the tissues.

Materials and Reagents

  1. Tissue specimens of small (terminal ileum) and large intestine
  2. RPMI 1640 (Life Technologies, catalog number: 21875-034 )
  3. Fetal Bovine Serum (FBS) (Sigma-Aldrich, catalog number: F7424 )
  4. HEPES (Life Technologies, Gibco®, catalog number: 15630-080 )
  5. Penicillin and Streptomycin (Life Technologies, catalog number: 15140-122 )
  6. HBSS (Life Technologies, catalog number: 14180-046 )
  7. EDTA (Life Technologies, catalog number: AM9261 )
  8. Liberase TM (Roche Diagnostics, catalog number: 05401127001 )
  9. DNase I (Sigma-Aldrich, catalog number: D4263 )
  10. Collagenase 1A (0.2 μm-filtered) (Sigma-Aldrich, catalog number: C9891 )
  11. FACS antibodies
    1. T cells
      CD3-PE-Cy7 (SK7) (BD biosciences)
      Pacific blue (PB)-CD8 (RPA-T8) (BD biosciences)
      Quantum dot (QD) 605-CD4 (S3.5) (Life Technologies, InvitrogenTM)
      LIVE/DEAD® Fixable Near IR Dead Cell Stain Kit (Life Technologies, InvitrogenTM)
    2. DC/macrophages
      CD3-PE-Cy5 (UCHT1) (eBioscience)
      CD19-PE-Cy5 (HIB19) (eBioscience)
      CD11c-PE-Cy7 (3.9) (eBioscience)
      CD103-PE (Ber-ACT8) or CD103-eFluor647 (B-Ly7) (eBioscience)
      CD14-eFluor450 (61D3) (eBioscience)
      HLA-DR-APCeFluor780 (L43) (eBioscience)
      CD20 PE-Cy5 (2H7) (BioLegend)
      TCRab-PE-Cy5 (IP26) (BioLegend)
      Biotin-or PE-Cy7 CD172a (SE5A5) (BioLegend)
      CD56-PE-Cy5 (Alpha Diagnostic Intl)
      Biotin- or FITC-CD11c (MJ4-27G12) (Miltenyi Biotec)
      CD141-PE (AD5-14H12) (Miltenyi Biotec)
      CD45 V500 (HI30) (BD Biosciences)
      Biotinylated antibodies were detected using streptavidin conjugated to PE-Cy7 (eBiosicence) or QDot605 (Life Technologies, InvitrogenTM).
      Dead cells were excluded from analysis using propidium iodide PI (Life Technologies, Molecular Probes®).
  12. R10 medium (see Recipes)

Equipment

  1. Cell strainer (100 μm) (Thermo Fisher Scientific, catalog number: 22363549 )
  2. 50 ml Falcon tube
  3. Polyester filters cut in 10 x 10 cm squares (mesh count 27 threads/cm, mesh opening 250 μm, thread diameter 120 μm) (Tekniska Precisionsfilter JR AB)
  4. Petri dish (SARSTEDT AG, catalog number: 82.1473 )
  5. Ultra low attachment 24-well culture plate (Sigma-Aldrich, catalog number: CLS3473 )
  6. Pipette
  7. Needle/forceps
  8. Surgical scissor/scalpel
  9. Beakers with lid (VWR International, catalog number: 216-2694 )
  10. 37 °C, 5% CO2 cell culture incubator
  11. Shaker
  12. Centrifuge
  13. Microscope
  14. Multicolour FACS analyser (BD Lsr II flow cytometer)

Software

  1. FlowJo software (Tree Star Inc)

Procedure

English
中文翻译

文章信息

版权信息

© 2014 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Uronen-Hansson, H., Persson, E., Nilsson, P. and Agace, W. (2014). Isolation of Cells from Human Intestinal Tissue. Bio-protocol 4(7): e1092. DOI: 10.21769/BioProtoc.1092.

ris ris Download Citation in RIS Format

分类

免疫学 > 免疫细胞分离 > 维持和分化

细胞生物学 > 细胞分离和培养 > 细胞分离

细胞生物学 > 细胞运动 > 细胞迁移

您对这篇实验方法有问题吗？

在此处发布您的问题，我们将邀请本文作者来回答。同时，我们会将您的问题发布到Bio-protocol Exchange，以便寻求社区成员的帮助。

0/150

tip 提问指南

+ 问题描述

写下详细的问题描述，包括所有有助于他人回答您问题的信息（例如实验过程、条件和相关图像等）。

post 发布问题
0 Q&A
pen 提交稿件