肾脏免疫细胞的可靠和高效率的提取
Summary
Techniques that are reliable and efficient for the isolation of kidney immune cells are needed for downstream applications. This requires surface antibody labeling of a small number of kidney immune cells. Herein, we describe a concise method for isolation of kidney immune cells that seemingly achieves this goal.
Abstract
Immune system activation occurs in multiple kidney diseases and pathophysiological processes. The immune system consists of both adaptive and innate components and multiple cell types. Sometimes, the cell type of interest is present in very low numbers among the large numbers of total cells isolated from the kidney. Hence, reliable and efficient isolation of kidney mononuclear cell populations is important in order to study the immunological problems associated with kidney diseases. Traditionally, tissue isolation of kidney mononuclear cells have been performed via enzymatic digestions using different varieties and strengths of collagenases/DNAses yielding varying numbers of viable immune cells. Recently, with the development of the mechanical tissue disruptors for single cell isolation, the collagenase digestion step is avoided and replaced by a simple mechanical disruption of the kidneys after extraction from the mouse. Herein, we demonstrate a simple yet efficient method for the isolation of kidney mononuclear cells for every day immune cell extractions. We further demonstrate an example of subset analysis of immune cells in the kidney. Importantly, this technique can be adapted to other soft and non-fibrous tissues such as the liver and brain.
Introduction
免疫系统激活发生在多个肾脏疾病和病理生理过程6,10,11,13。活跃的研究的潜在领域包括对免疫系统的激活的各种触发器，各种细胞类型涉及，在一个特定的疾病设置的细胞因子/趋化因子的模式，由特定的药物等。所有前述过程的调制为了举例说明，在缺血 – 再灌注损伤（急性肾损伤的模型），有增加的免疫细胞或骨髓衍生的造血细胞或CD45 +细胞在几个小时内，这是通过维修或纤维化的期间持续（6周后）5， 12。这些免疫细胞分泌促炎性和抗炎性细胞因子和趋化因子既来编排修复5,12的过程。目前，同时使用多个荧光团来标记细胞群体中的单个细胞悬浮液的能力已经与广告增加发泄现代流式细胞仪机与四到五激光器。这大大增加的能力来区分基于其功能状态3,7的细胞群。例如，为了精确地标记巨噬细胞为低 ，将在相同的样品体积是需要的栅极F4 / 80 低的CD11b 高 Ly6b 高 CD206至少3个荧光团为活细胞，CD45 +（白细胞）和Ly6G-（中性粒细胞）和这是与新的流式细胞仪3很可能的。然而，对于细胞因子的分泌，细胞增殖，细胞毒性，巨噬细胞活化和淋巴细胞和单核细胞的各种子集的数目的量化的下游分析不仅需要有良好的质量（活细胞，单峰），但细胞的足够数量。
在肾脏的免疫系统由两个适应性和先天组件和多种细胞类型1,7,13的。例如，在小鼠中两个小子分离（1.4×10 6个细胞）和它们的约5-15％（1,400-4,200）的总肾免疫细胞neys一起被报道含有2-17％（28,000-266,000） 的 CD45 +细胞是CD4 +细胞1 ，5,12。这些CD4 +细胞的一小部分（5-15％，70-630）是FOXP3 +细胞（ 图1）1。由于在细胞中的百分比这些步骤明智的减少，有时感兴趣的细胞群（在这种情况下，CD45 + CD4 +的FoxP3 +细胞）是由单纯的〜100个细胞表示。的CD45 + CD4 +的FoxP3 +细胞的数量少，因此必须有大量的总细胞分离并在细胞是质量好于下游的研究，例如细胞因子分泌测定。此外，它可能是必要的，因为亚群中不足够高的数字表示来执行量化测定以肾脏从2-3只小鼠相结合。因此，肾单核细胞群体的可靠和有效的隔离是可取的，以便螺柱y随肾脏疾病相关的免疫学光谱。
传统上，肾单核细胞的分离，研究人员使用了多种酶消化，如胶原酶1A或II包括DNA酶1 1,5,12的。它公知的是胶原酶具有与批号和由公司制造的不同而不同，因此有必要滴定为最佳浓度和温育4,14,15的持续时间的酶活性。此外，用胶原酶消化添加时间为切碎肾成小块，就必须肾脏条在EDTA中加热（37℃）浴和额外的时间为孵育的孵育终止反应。此外，较少的不育性可以针对某些下游程序需要的细胞培养来实现。更重要的是，根据所涉及的研究者和所有的变量，它会导致变异跨实验室数据和解释。近年来，随着机械组织干扰/均质16的发展，胶原酶消化步骤是完全可以避免和肾脏2的简单机械破碎代替。在此，我们展示了肾脏免疫细胞的日常免疫细胞提取隔离一个简单而有效的方法。重要的是，这种技术可以适用于其他柔软和非纤维组织如肝和脑16。
Protocol
Representative Results
Discussion
我们在这里提出了一个方法，以获得从肾脏的免疫细胞中的可靠和有效的方式。主要的修改的广泛使用的胶原酶消化步骤（组织机械破碎）保存约30分钟，并有大量活的免疫细胞的隔离下两个小时4肾脏样品花费。此外，根据我们的研究问题，我们现在只用一个单一的肾（其他肾脏可以用于通过Western印迹，免疫组织化学和mRNA分析通过PCR蛋白质分析）对我们的免疫细胞的分离和常规获得〜2×10 6</…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work is supported by a Research Grant from Dialysis Clinics Inc. and from the University of Missouri Research Board Grant.
Materials
| Stomacher 80 Biomaster lab system | Seward | ||
| Stomacher 80 Classic bags | Seward | BA6040/STR | |
| Sorvall Legend XFR Centrifuge | Thermo Scientific | Or equivalent equipment | |
| Hemocytometer | Electron Microscopy Sciences | 63514-11 | |
| Analytical flow cytometer | BD LSR-X20 Fortessa | ||
| Percoll | Sigma | P1644 | |
| Dulbecco’s phosphate buffered saline 1X (DPBS) | Gibco, Life Technologies | 14190-250 | |
| Polypropylene tubes, no cap | Becton Dickinson | 352002 | |
| Fixable Viability Stain | BD Biosciences | FVS510, 564406 | |
| Anti-CD16/32 (Clone: 93) | EBioscience | 14-0161 | |
| anti-CD45 (clone: 30-F11) BV421 | BD Pharmingen | 103133/4 | |
| Anti-Foxp3 (Clone: FJK-16s) APC | EBioscience | 17-5773 | |
| Anti-CD127 (Clone: A7R34) PE/Cy7 | Biolegend | 135013/4 | |
| anti-CD44 (Clone: IM7) PerCP/Cy5.5 | Biolegend | 103031/2 | |
| anti-CD4 (Clone: RM4-5) APC-Cy7 | Biolegend | 100413/4 | |
| anti-CD8 (Clone: 53-6.7) BV785 | Biolegend | 100749/50 | |
| Anti-Ly6G (Clone: 1A8) FITC | Biolegend | 127605/6 | |
| Anti-CD11b (Clone: M1/70) PerCP-Cy5.5 | Biolegend | 101227/8 | |
| Anti-F4/80 (Clone: BM8) APC | Biolegend | 123115/6 | |
| Anti-CD11c (Clone: N418) BV785 | Biolegend | 117335/6 | |
| Anti-CD301 (Clone: LOM-14) PE-Cy7 | Biolegend | 145705/6 | |
| Anti-CD26 (Clone: H194-112) PE | Biolegend | 137803/4 | |
| 100 μm filter | Fisher Scientific | 22363548 | |
| Fisherbrand Tubes 50 ml | Fisher | Or equivalent equipment | |
| Fisherbrand Tubes 15 ml | Fisher | Or equivalent equipment | |
| Sucrose | Fisher chemical | S5-3 | |
| Transfer pipette fine tip | Samco Scientific | 232 | Or equivalent equipment |
| Flow Cytometery Staining Buffer Solution | EBioscience | 00-4222-26 | Or equivalent equipment |
| 1X RBC Lysis Buffer | EBioscience | 00-4333-57 | Or equivalent equipment |
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