从肾脏双重否定αβ性T细胞的分离
Summary
DNabT细胞是罕见的外周T细胞之间;然而，他们是丰富的在某些非淋巴组织。从非淋巴组织中分离的DN T细胞的难度阻碍了其功能的分析尽管越来越多的认可的病理生理意义。我们描述了一种新的协议，用于从小鼠肾脏高度纯化的DN T细胞的隔离。
Abstract
目前还没有标准协议，用于DN T细胞从非淋巴组织的隔离，尽管它们在不同的免疫应答日益报道的参与。 DN T细胞是有牵连的调 节免疫和自身免疫应答和耐受allotransplants 1-6独特的免疫细胞类型。然而DN T细胞，罕见的外周血和次级淋巴器官（脾脏和淋巴结），但都在正常肾脏的主要居民。很少有人知道他们的病理生理功能7，由于其在外围缺乏。最近，我们在稳定状态，并在缺血再灌注损伤中描述的肾脏8这部分人口的综合表型和功能分析。的DN T细胞功能分析将通过开发一个协议，用于从肾脏的隔离大大提高。
在这里，我们描述了一种新的协议，允许isolatioÑ高纯度AB的CD4 + CD8 + T细胞和DN T细胞的小鼠肾脏。简单地说，我们用胶原酶消化肾组织，并通过密度梯度分离肾单核细胞（KMNC）。这是由两个步骤，从KMNC丰富的造血T细胞从3％至70％。第一步骤包括一个正选择使用CD45阳性分离试剂盒的造血细胞。在第二步骤中，DN T细胞被除去使用CD4，CD8，和MHC II类的单克隆抗体和CD1d的α-GalCer的四聚体的非期望的细胞的负分离。这个策略导致了90％以上的纯DN T细胞群。表面染色用上述抗体随后通过FACS分析用于确定纯度。
Introduction
周边αβTCR+ CD3 + CD4 – CD8 –双阴性（DN）T细胞被分成具有不同的表型和功能1-4各种子集。 DN T细胞知之甚少，但越来越多地被牵连在不同的疾病模型4-6病理免疫反应。
DN T细胞，是在各种免疫和自身免疫应答的调节和异体移植耐受的调制日益牵连了独特的免疫细胞类型。4-6，9它们是罕见的外周血和次级淋巴器官（脾和淋巴结）。然而，它们的正常肾和肠上皮10-12主要居民。很少有人知道他们在稳定状态功能7在肾和病理条件下，如肾transpl相关的急性肾损伤（AKI）下蚂蚁。
因为不同的免疫细胞在调节免疫反应，包括alloresponses，限定每个游戏者的角色的错综复杂的角色是在理解alloresponses和设计新疗法至关重要。给出的显著号码存在于生理和不同疾病状态下的肾DN T细胞，DN的T细胞很可能在移植受者起到在小鼠和人类调节免疫和自身免疫应答的关键作用，并且alloresponses。积累虽然仍散落证据牵连的DN T细胞在致病性和免疫功能，但它是知之甚少，为什么，以及他们如何表现出特定的有害或抑制功能和环境如何影响他们。
由于它们的低丰度在肾脏，分离的改进的方法是必要的。
目前还没有标准协议，用于从T DN T细胞的分离他非淋巴组织。我们的协议描述了一种新颖的方法，用于从肾脏DN T细胞的隔离;然而，也可用于各种非淋巴组织的方法。
Protocol
Representative Results
Discussion
有一个在DN T细胞增加的兴趣，因为他们被牵连在不同的病理情况，如自身免疫性疾病，癌症，移植耐受和肾脏，包括急性肾损伤（AKI）的原发疾病，肾小球肾炎8，13。因此，有必要更好地理解和描述的DN T细胞的病理生理功能。然而，目前仍缺乏这些细胞的功能相比，CD4和CD8 T细胞的了解。一个主要的原因是DN T细胞在次级淋巴器官的缺乏，因此难以取得足够的细胞在体外分析和过继…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项工作是由美国国立卫生研究院的PBS（R21 AI095484）的支持。我们感谢健保四聚体核心设施的CD1d四聚体。
Materials
| Laminar flow hood | Baker Company, Inc | Or equivalent equipment | |
| Centrifuge | Beckman Coulter | Or equivalent equipment | |
| Hemocytometer | Electron Microscopy Sciences | 63514-11 | |
| Atmosphere-controlled incubator | Fisher Scientific | (37°C with 5% CO2) | |
| Microscope | Olympus | Or equivalent equipment | |
| Analytical flow cytometer | LSR II | ||
| Name of the reagent | Company | Catalog Number | Comments |
| RPMI 1640 | Media Tech | 10-040-CV | |
| Collagenase D | Roche | 11088858001 | |
| Percoll | GE Healthcare | 17-0891-01 | |
| Fetal bovine serum | Corning Cellgro | 35-011-CV | |
| 0.1% sodium azide (NaN3) | Sigma-Aldrich | S2002 | |
| Buffer phosphate buffered saline (PBS) | Corning Cellgro | 21-040-CV | |
| PBS 10X | Mediatech | 46-013-CM | |
| EDTA buffer | Sigma-Aldrich | E1161 | |
| LS columns | MiltenyiBiotec | 130-042-401 | |
| CD45+ microbeads | MiltenyiBiotec | 6.78E-51 | |
| Biotin microbeads | MiltenyiBiotec | 130-090-485 | |
| anti-CD45 (clone: 30-F11) PerCP | eBioscience | G1397 | |
| anti-CD45 (clone: 30-F11) APC-Cy7 | Biolegend | 103116 | |
| anti-TCR Pacific Blue (ab-chain, clone: H57-597) | Invitrogen | HM3628 | |
| anti-CD4 PE | eBioscience | 12-0043 | |
| anti-CD8 FITC | eBioscience | 8011-0087 | |
| anti-CD4 biotinylated (GK1.5) | BD | 553728 | |
| anti-CD8 biotinylated (clone 53-6.7) | BD | 553029 | |
| anti-Fc receptor (CD16/32) biotinylated | BD | 553143 | |
| anti-MHC class II (I-A d) biotinylated | BD | 553609 | |
| anti-CD1d PBS-57 tetramer | NHI tetramer | lote # 15621-PBS57 | |
| AutoMACS Running Buffer- MACS Separation Buffer | MIlteny I Biotec | 130-091-221 | |
| C57BL/6J mice | Jackson Labs | 000664 | Kidneys – Lymph Nodes |
| Petri dish | BD Biosciences | 356517 | |
| 70mc filter | Fisher Scientific | 22363548 | |
| Plunger | Or equivalent equipment | ||
| GeneMate Tubes 50 ml | Bioexpress | C-3394-3 | Or equivalent equipment |
| GeneMate Tubes 15 ml | Bioexpress | C-3394-1 | Or equivalent equipment |
| Petri Dish | Fisher Scientific | S33580 | |
| Plate 24 wells | BD Biosciences | 354723 |
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