考试流式细胞仪检测胸腺阳性和阴性选择
Summary
我们提出了一种基于流式细胞仪的方法来研究T细胞发育<em在体内</em使用基因操纵的野生型小鼠的T细胞受体的转基因背景。
Abstract
健康的免疫系统需要对外来抗原的T细胞反应，而其余的宽容，以自身抗原。随机重排的T细胞受体（TCR）的α和β位点产生一个具有丰富的多样性，在抗原特异性T细胞库，无论是对自己和国外。安全和有用的T细胞在胸腺发育过程中的曲目的选择是至关重要的。胸腺选择的缺陷的发展，自身免疫性疾病和免疫缺陷病的1-4。
T细胞祖细胞进入胸腺双阴性（DN）胸腺细胞不表达CD4或CD8共受体。 αβTCR和两个共受体的表达发生在双阳性（DP）阶段。互动的自我肽-MHC（住房抵押贷款公司）提出的胸腺细胞的αβTCR确定DP胸腺细胞的命运。高亲和力相互作用导致负选择和消元重刑自反应的胸腺细胞。在积极的选择和发展的CD4或CD8单阳性（SP）T细胞能够识别外来抗原的自我MHC 5低亲和力相互作用的结果。
的阳性筛选可以研究在小鼠的多克隆（野生型）的TCR剧目通过观察成熟T细胞的产生。然而，他们研究的阴性选择，这涉及到删除的小抗原特定人群的理想选择。许多模型系统已被用于研究的阴性选择，但不同的能力复述的生理活动6。例如， 在体外刺激的胸腺细胞缺乏选择是密切参与胸腺环境，而施用外源性抗原可导致非特异性缺失胸腺细胞7-9。目前，最好的工具，研究在体内的负选择，表达一个transg的有老鼠ENIC TCR特异的内源性自身抗原。然而，许多经典的TCR转基因模型的特点是在DN阶段的过早表达的转基因TCRα链，导致过早的负选择。我们的实验室已经开发出了HY的CD4模型，其中转基因HY，TCRα是有条件表示在自行分配学位阶段，让阴性选择过程中发生的DP，SP转型发生在野生型小鼠10。
在这里，我们描述了一个基于流式细胞仪的协议在HY CD4小鼠模型，研究胸腺的阳性和阴性选择。虽然负选择HY CD4小鼠中是高度生理，这些方法还可以被应用到其他的TCR转基因模型。我们也将目前的一般策略分析正选择一个适用于任何转基因操作的小鼠多克隆剧目。
Protocol
请参阅图1为实验方案的总体方案。 1。解剖地方无菌钢丝网屏幕为60 x 15 mm的培养皿中。需要一个单元的每个组织样本。 每个培养皿中加入5ml的Hank氏平衡盐溶液（HBSS）。保持在冰上菜。 安乐死小鼠与CO 2。 安全小鼠剥离面，腹一面朝上。用70％乙醇进行杀菌，并确保皮草纠结向下喷雾鼠标。 用手术剪，开始清扫腹切口?…
Representative Results
在生理TCR转基因模型和野生型小鼠，正开始选择在DP 明亮的舞台前，后移动到DP 沉闷的阶段抗原相遇。 DP 沉闷的胸腺细胞，然后进入一个过渡CD4 + CD8 罗的舞台前，成为CD4SP或CD8SP胸腺细胞（ 图2B）。成熟的SP胸腺细胞的特点是高的TCR表达和损失的CD24（ 图2C）。虽然CD8 CD4配置文件可以发现缺陷，积极的选择，检查TCRβCD69或CD5可以提?…
Discussion
这里提出的协议可以被用来检查非TCR转基因和TCR转基因小鼠中的阳性和阴性选择。此协议描述了表面抗原染色。有关的分子机制的进一步的分析，这是经常需要进行细胞内染色。我们使用了BD Biosciences公司的Cytofix / Cytoperm工具包的大多数细胞内的蛋白质和BD Biosciences公司的Foxp3的染色试剂盒的转录因子。通常，我们获得了我们的样品后，立即染色。然而，样品可以被存储在FACS缓冲液中过夜用1％多?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
的作者想感谢张冰，他的技术援助。这项工作是由加拿大卫生研究院的研究（MOP-86595）。 TAB是一个CIHR的新研究者和AHFMR学术。 QH的支持，一个CIHR加拿大研究生奖学金 – 博士和一个AIHS的全日制学籍。 SAN是由英国女王伊丽莎白二世研究生奖学金的支持。 AYWS支持由NSERC – 博士研究生奖学金。
Materials
| Name of the reagent | Company | Catalogue number | Comments (optional) |
| HyClone Hank’s balanced salt solution | Thermo Scientific | SH30030.02 | |
| Metal mesh screens | Cedarlane | CX-0080-E-01 | |
| Petri dishes (60 x 15 mm) | Fisher Scientific | 877221 | |
| Syringes (3 ml) | BD Biosciences | 309657 | |
| Conical tubes (15 ml) | Sarstedt | 62.554.205 | |
| Microscope | Zeiss – Primo Star | 415500-00XX-000 | |
| Hemocytometer | Hausser Scientific | 3110 | |
| 96-well plate | Sarstedt | 82.1582.001 | |
| Multichannel pipette | Fisherbrand | 21-377-829 | |
| Fetal calf serum | PAA | A15-701 | |
| Phosphate buffered saline | Fisher Scientific | SH3025802 | |
| Sodium azide | IT Baker Chemical Co. | V015-05 | |
| FcR blocking reagent | Clone 2.4G2 | ||
| Anti-mouse HY TCR | eBioscience | XX-9930-YY* | Clone T3.70 |
| Anti-mouse CD4 | eBioscience | XX-0042-YY* | Clone RM4-5 |
| Anti-mouse CD8α | eBioscience | XX-0081-YY* | Clone 53-6.7 |
| Anti-mouse CD24 | eBioscience | XX-0242-YY* | Clone M1/69 |
| Anti-mouse TCRβ | eBioscience | XX-5961-YY* | Clone H57-597 |
| Anti-mouse CD69 Biotinylated | eBioscience | 13-0691-YY* | Clone H1.2F3 |
| Anti-mouse CD5 Biotinylated | eBioscience | 13-0051-YY* | Clone 53-7.3 |
| Streptavidin | eBioscience | XX-4217-YY* | |
| Flow cytometer | BD Biosciences – FACS Canto | 338962 | |
| FACS tubes | BD Biosciences | 352052 | |
| Flow cytometry analysis software | TreeStar – Flowjo | FlowJo v7/9 | |
| HyClone RPMI – 1640 medium | Thermo Scientific | SH30027.01 | |
*XX varies by fluorochrome and YY varies by vial size. |
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Tags
Thymic Positive SelectionThymic Negative SelectionFlow CytometryT Cell Receptor (TCR)Antigen SpecificitySelf-antigensT Cell RepertoireAutoimmune DisordersImmunodeficiency DisordersDouble Negative (DN) ThymocytesDouble Positive (DP) StageCD4 Co-receptorCD8 Co-receptorSelf-peptide-MHC (pMHC)High Affinity InteractionsLow Affinity InteractionsPositive SelectionSingle Positive (SP) T CellsForeign AntigensPolyclonal TCR Repertoire
Cite This Article
Hu, Q., Nicol, S. A., Suen, A. Y., Baldwin, T. A. Examination of Thymic Positive and Negative Selection by Flow Cytometry. J. Vis. Exp. (68), e4269, doi:10.3791/4269 (2012).