小鼠小肠淋巴细胞的分离和流式细胞表征
Summary
There is growing interest in the quantitative characterization of intestinal lymphocytes owing to increasing recognition that these cells play a critical role in a variety of intestinal and systemic diseases. In this protocol, we describe how to isolate single cell populations from different small-intestinal compartments for subsequent flow cytometric characterization.
Abstract
肠子 – 包含在身体任何器官的免疫细胞的数量最多 – 经常暴露于外来抗原，无论是微生物和饮食。鉴于越来越理解是，这些管腔抗原帮助塑造免疫反应和免疫细胞的教育中肠为数字全身性疾病的关键，已经在表征肠道免疫系统的兴趣增加。然而，许多已发表的协议是艰苦和耗时的。我们在这里提出一个简化的协议用于从小肠固有，上皮内层，和淋巴集结是快速的，可重复，并且不需要费力的Percoll梯度淋巴细胞的隔离。虽然该协议着重于小肠，它也适用于结肠的分析。此外，我们突出一些方面中，可能需要额外的优化取决于特定科学疑问句化。这种方法导致了大量可行的淋巴细胞，可以随后用于流式细胞术分析或表征备用装置的隔离。
Introduction
小肠的主要任务通常被认为是营养物1的消化和吸收。虽然这种代谢功能显然必不可少的，小肠具有在保护宿主免受内腔2内发现的环境抗原的持续拦河坝同样显著作用。肠道隔开外界（ 例如 ，管腔抗原）从主机的使用本身是只有一个单一的电池层厚的上皮细胞层的内部环境。这样，小肠道免疫系统具有平衡其为反应性的阈值，允许从饮食和共生微生物外来抗原以最小的进入黏膜，如果有的话，免疫反应而安装对抗入侵的病原体和强大的响应的艰巨任务其他“有害的”抗原。到这些抗原过度或不适当的免疫应答可导致病理疾病（ 如 ，inflamma托里肠炎，I型糖尿病，多发性硬化），必须避免3-6。
总体而言，在胃肠道被认为代表在体内最大的免疫器官，含有在所有抗体分泌细胞7的70％。小肠道免疫系统由3个主要的隔层-固有层（LP）时，上皮内层和Peyer氏斑（PPS） -每个包含淋巴细胞2的独特基团。在LP淋巴细胞（LPLs）主要TCRαβ+ T细胞以〜20％的B细胞;上皮内淋巴细胞（IELs）包含极少的B细胞与更多的TCRγδ+ T细胞比TCRαβ+ T细胞;和PPS，它们是嵌入在小肠壁次级淋巴器官，包含〜80％的B细胞。虽然这些解剖区域的稍有不同的功能和本体论基础，他们在发挥作用啊armonized时尚，以防止致病性侮辱的主机。
此外，有越来越多的赞赏菌群对肠道免疫系统的发展的关键因素，随着识别特定的微生物和特定的细胞谱系8,9的个体发育之间的同源关系。此外，鉴于肠道免疫系统的教育影响解剖学远处的免疫应答（ 例如 ，关节炎，多发性硬化症，肺炎），它已经很清楚，在肠道免疫系统的发展是有关多疾病过程比先前识别10 -12。这样，在定量评估肠道免疫系统的兴趣已扩展超出宿主 – 病原体相互作用现在包括主机共生相互作用和许多全身性疾病的发病机制，以及。
定在当前的方法的可变性肠淋巴细胞隔离，即对产量，活力，和一致性优化同时平衡所需要的时间是越来越重要的方法。涉及的Percoll梯度协议是时间和劳动力密集型的，而且可能更容易出现人为错误，导致可变的产量和存活率13。在此，我们提供了淋巴细胞的所有3小肠道免疫车厢分离和鉴定优化的协议。此外，在粘膜免疫系统微生物引起的改变给出越来越大的兴趣，我们包括可用于允许微生物小鼠之间的水平传播，以评估这些变化如何定量影响肠道免疫系统的步骤。
Protocol
Representative Results
Discussion
我们提出了一个协议，用于分离和流动的小肠淋巴细胞，包括LPLs，IELs，并在PPS淋巴细胞术表征。对于那些有兴趣在评估中微生物的变化如何影响小肠的免疫系统，我们详细介绍参与小鼠窝藏不同microbiotas之间的生物体的水平传播的简单步骤。虽然该协议的重点是小肠，该过程是用于大肠的分析是相同的，唯一的区别是，有没有的PP去除（尽管结肠补丁都存在，这些通常不是严重可见）。
<p class…Divulgations
The authors have nothing to disclose.
Acknowledgements
NKS is supported by NIH award K08 AI108690.
Materials
| Sterile Gloves | Kimberly-Clark | 555092 | |
| sterile mouse cage | Innovive | MS2-AD | contains lid, cage bottom, and alpha-dri bedding |
| metal feeder | Innovive | M-FEED | |
| water bottle | Innovive | M-WB-300 | |
| card holder | Innovive | CRD-HLD-H | |
| autoclavable rodent chow (NIH-31M) | Zeigler | 4131207530 | |
| RPMI medium 1640 | Gibco | 11875-119 | |
| dithiothreitol (DTT) | Sigma | D5545-5G | |
| 0.5 M EDTA (pH 8.0) | Ambion | AM9262 | |
| fetal bovine serum (FBS) | GemBio | 100-510 | |
| dispase II | Invitrogen | 17105-041 | the concentration in the protocol is based on an activity level of 1.878 U/mg |
| collagenase, type II | Invitrogen | 17101-015 | the concentration in the protocol is based on an activity level of 245 U/mg |
| dissecting scissors | Roboz | RS-5882 | |
| feeding needle (18 G, 2" length) | Roboz | FN-7905 | |
| 10 ml syringe | BD | 305482 | |
| PBS | Gibco | 14190-250 | |
| Disposable Scalpel (15 blade) | Miltex | 4-415 | |
| curved forceps | Roboz | RS-5211 | |
| straight forceps | Roboz | RS-5132 | |
| multi-purpose cups, 120 ml | VWR | 89009-662 | |
| stir bar | VWR | 58949-062 | |
| multi-position stir plate, 9-position | VWR | 12621-048 | |
| stainless steel conical strainer, 3 inch | RSVP | ||
| 1.5 ml tube | Eppendorf | 0030 125.150 | |
| 100 μm cell strainer | Falcon | 08-771-19 | |
| 40 μm cell strainer | Falcon | 08-771-1 | |
| 50 mL conical tube | Falcon | 352098 | |
| 1 ml syringe | BD | 309659 | |
| 96-well plate, round-bottom | Corning | 3799 | |
| anti-mouse CD16/32 (Fc block) | Biolegend | 101320 | |
| (optional) fixable viability dye eFluor 780 | eBiosciences | 65-0865-18 | |
| 10% formalin, neutral buffered | Thermo Scientific | 5725 |
References
- Cummings, D. E., Overduin, J. Gastrointestinal regulation of food intake. J Clin Invest. 117 (1), 13-23 (2007).
- Mowat, A. M., Agace, W. W. Regional specialization within the intestinal immune system. Nat Rev Immunol. 14 (10), 667-685 (2014).
- Round, J. L., Mazmanian, S. K. The gut microbiota shapes intestinal immune responses during health and disease. Nat Rev Immunol. 9 (5), 313-323 (2009).
- Sartor, R. B. Microbial influences in inflammatory bowel diseases. Gastroenterology. 134 (2), 577-594 (2008).
- Tlaskalova-Hogenova, H., et al. Commensal bacteria (normal microflora), mucosal immunity and chronic inflammatory and autoimmune diseases. Immunol Lett. 93 (2-3), 97-108 (2004).
- Wen, L., et al. Innate immunity and intestinal microbiota in the development of Type 1 diabetes. Nature. 455 (7216), 1109-1113 (2008).
- Pabst, R., Russell, M. W., Brandtzaeg, P. Tissue distribution of lymphocytes and plasma cells and the role of the gut. Trends Immunol. 29 (5), 206-208 (2008).
- Surana, N. K., Kasper, D. L. The yin yang of bacterial polysaccharides: lessons learned from B. fragilis PSA. Immunol Rev. 245 (1), 13-26 (2012).
- Surana, N. K., Kasper, D. L. Deciphering the tete-a-tete between the microbiota and the immune system. J Clin Invest. 124 (10), 4197-4203 (2014).
- Gauguet, S., et al. Intestinal microbiota of mice influences resistance to Staphylococcus aureus pneumonia. Infect Immun. , (2015).
- Ochoa-Reparaz, J., et al. A polysaccharide from the human commensal Bacteroides fragilis protects against CNS demyelinating disease. Mucosal Immunol. 3 (5), 487-495 (2010).
- Wu, H. J., et al. Gut-residing segmented filamentous bacteria drive autoimmune arthritis via T helper 17 cells. Immunity. 32 (6), 815-827 (2010).
- Goodyear, A. W., Kumar, A., Dow, S., Ryan, E. P. Optimization of murine small intestine leukocyte isolation for global immune phenotype analysis. J Immunol Methods. 405, 97-108 (2014).
- Chung, H., et al. Gut immune maturation depends on colonization with a host-specific microbiota. Cell. 149 (7), 1578-1593 (2012).
- Resendiz-Albor, A. A., Esquivel, R., Lopez-Revilla, R., Verdin, L., Moreno-Fierros, L. Striking phenotypic and functional differences in lamina propria lymphocytes from the large and small intestine of mice. Life Sci. 76 (24), 2783-2803 (2005).
- Carrasco, A., et al. Comparison of lymphocyte isolation methods for endoscopic biopsy specimens from the colonic mucosa. J Immunol Methods. 389 (1-2), 29-37 (2013).
- Van Damme, N., et al. Chemical agents and enzymes used for the extraction of gut lymphocytes influence flow cytometric detection of T cell surface markers. J Immunol Methods. 236 (1-2), 27-35 (2000).
