制备髓源性抑制细胞从幼稚和胰腺癌荷瘤小鼠，用流式细胞仪和自动磁性细胞分选（AutoMACS）（MDSC）
Summary
这是一个迅速和全面的免疫髓源性抑制细胞（MDSC）和丰富GR-1的方法<sup> -</sup>从小鼠脾脏白细胞。这种方法使用流式细胞仪和AutoMACS细胞排序丰富可行的GR-1<sup> -</sup>白细胞之前使用的MDSC排序，以流式细胞仪<em>在体内</em>和<em>在体外</em>检测。
Abstract
的MDSC是一个不成熟的巨噬细胞，树突状细胞和粒细胞积聚在病理条件下的淋巴器官，包括寄生虫感染，炎症，创伤后应激，移植物抗宿主病，糖尿病和癌症1-7异构人口。在小鼠的MDSC明示的Mac-1（细胞CD11b）和GR-1（Ly6G和Ly6C）表面抗原。重要的是要注意的MDSC荷，它们显着扩大在各个主机研究和压制7-10天真对口相比抗肿瘤免疫反应。然而，根据病理状态，有不同的MDSC亚与不同的机制和抑制11,12的目标。因此，有效的方法来隔离可行的MDSC人口是重要的，在阐明各自不同的分子机制抑制在体外和体内 。
近日，在Ghansah组报告的MDSC在小鼠胰腺癌模型的扩展。我们的肿瘤轴承的MDSC显示动态平衡的损失，并增加抑制功能比较幼稚的MDSC 13。的MDSC的百分比显着天真与荷瘤小鼠的淋巴车厢。这是一个重大的警告，这往往阻碍了这些的MDSC准确的比较分析。因此，丰富天真小鼠GR-1 +白细胞荧光激活细胞分选（FACS）前提高纯度，活力和排序的时间显着降低。然而，丰富的GR-1 +荷瘤小鼠的白细胞是可选的，因为这些丰富的排序快速流式细胞仪。因此，在这个协议中，我们描述了一个高效的方法，免疫的MDSC从排序的MDSC及时天真小鼠脾脏和丰富的Gr-1 +白细胞。免疫C57BL / 6小鼠接种小鼠Panc02 CE的LLS皮下而天真的小鼠接受1XPBS。大约30天接种后脾脏被采伐和加工成单细胞悬液采用细胞分离筛。脾然后红细胞（RBC）的裂解，这些白细胞等分对MAC-1和GR-1免疫的MDSC百分比用流式细胞仪使用荧光标记抗体染色。在一个类似的实验，从整个天真的小鼠白细胞染色与荧光标记的GR-1抗体，培养与PE的微珠和正选择使用一个自动化的磁性活化细胞分选（autoMACS）分离。接下来，GR-1 +白细胞等份与Mac-1抗体染色，以确定在使用流式细胞仪的MDSC百分比增加。现在，这些GR1 +丰富的白细胞是准备用于比较分析（天真与荷瘤） 在体内和体外的MDSC排序流式细胞仪</em>的检测。
Protocol
开始之前，请准备以下解决方案： 3％，染色媒体（SM）： -3％的胎牛血清（FBS），1X的磷酸盐缓冲液（PBS） 磁珠缓存（MB）： – 牛血清白蛋白（BSA）的0.5％白蛋白在1XPBS 1。收获从小鼠脾脏皮下注入1.5×10 5小鼠Panc02细胞悬浮在100μL1X PBS 6-8周岁的C57BL / 6…
Discussion
这是一个详细的方法处理和immunophentyping的MDSC人口，是适用于各种动物模型不同的淋巴组织。特别，autoMACS富集，可用于各种白细胞的人口，包括GR-1枯竭脾4，从脾和淋巴结肿大5粒细胞亚群的净化，分 离骨髓中性粒细胞14和纯化的CD8 + T细胞从脾隔离和淋巴结15。无论所需的淋巴组织产生白细胞的单细胞悬液的细胞群体的利益，需要最佳表面染色，流式?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们承认USF流式细胞仪核心设施。我们想感谢丹尼斯库珀博士资源共享。我们也想感谢他们的协助玛雅科恩，劳拉·彭德尔顿和戴安娜拉图尔建立和拍摄这部影片。神经网络的支持NSF FG-LSAMP桥博士奖学金人力资源开发＃0929435。这项工作是由美国癌症学会院校研究资助＃93-032-13/Moffitt癌症中心颁发到TG。
Materials
| REAGENT | COMPANY | CATALOG # | COMMENTS |
| 1X Phosphate Buffered Saline | Thermo Scientific Hyclone | SH30028.02 | Ca2+/Mg2+/Phenol Red-free |
| Albumin from Bovine Serum (BSA) | Sigma-Aldrich | A7906 | Let BSA dissolve undisturbed in PBS; Sterile Environment |
| Fetal Bovine Serum (FBS) | Thermo Scientific Hyclone | SV3001403HI | Heat Inactivated; Sterile Environment |
| Rat anti-mouse CD16/32 monoclonal antibody (Fc Block) | BD Biosciences | 553142 | Sterile Environment |
| Anti-mouse CD11b (Mac-1) FITC | eBiosciences | 11-0112 | Sterile Environment |
| Anti-mouse Ly6G (Gr-1) APC | eBiosciences | 17-5931 | Sterile Environment |
| Anti-mouse Ly6G (Gr-1) PE | eBiosciences | 12-5931 | Sterile Environment |
| DAPI | Invitrogen | D1306 | Serial Dilution Sterile Environment |
| Cell Dissociation Sieve | Sigma-Aldrich | CD1-1KT | Autoclave before use |
| 70-μm strainer | BD Biosciences | 352350 | Sterile Environment |
| 1X RBC Lysis Buffer | eBiosciences | 00-4333-57 | Warm to room temperature before use; Sterile Environment |
| Petri dishes | Fisher Scientific | 08-757-12 | Sterile Environment |
| 50ml conical tubes | Thermo Scientific | 339652 | Sterile Environment |
| 5ml 12X75mm polystyrene round bottom tubes | BD Biosciences | 352054 | Known as FACS tubes; Sterile Environment |
| 96-well V-bottom plates | Corning | 3897 | Sterile Environment |
| Trypan Blue | Cellgro | 25-900-CI | Sterile Environment |
| PE MicroBeads | Miltenyi Biotec | 130-048-801 | Sterile Environment |
| AutoMACS Pro Separator | Miltenyi Biotec | 130-092-545 | |
| AutoMACS Columns | Miltenyi Biotec | 130-021-101 | |
| AutoMACS Running Buffer | Miltenyi Biotec | 130-091-221 |
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Tags
Myeloid Derived Suppressor Cells (MDSC)Naive MicePancreatic Tumor-bearing MiceFlow CytometryAutomated Magnetic Activated Cell Sorting (AutoMACS)Heterogeneous PopulationImmature MacrophagesDendritic CellsGranulocytesLymphoid OrgansParasitic InfectionInflammationTraumatic StressGraft-versus-host DiseaseDiabetesCancerMac-1 (CD11b)Gr-1 (Ly6G And Ly6C)Tumor-bearing HostsAnti-tumor Immune ResponsesSubpopulations Of MDSCMolecular Mechanisms Of SuppressionGhansah GroupMurine Pancreatic Cancer ModelHomeostasisSuppressive FunctionLymphoid CompartmentsGr-1+ Leukocytes
Cite This Article
Nelson, N., Szekeres, K., Cooper, D., Ghansah, T. Preparation of Myeloid Derived Suppressor Cells (MDSC) from Naive and Pancreatic Tumor-bearing Mice using Flow Cytometry and Automated Magnetic Activated Cell Sorting (AutoMACS). J. Vis. Exp. (64), e3875, doi:10.3791/3875 (2012).