三维人体免疫细胞可视化的光片显微术
Summary
在这里, 我们提出了一个协议, 以可视化的免疫细胞嵌入在三维 (3D) 胶原基质中使用光片显微镜。该协议还阐述了如何在3D 中跟踪单元迁移。该协议可用于3D 矩阵中其他类型的悬浮单元。
Abstract
在体内, 免疫细胞的活化、增殖和功能都发生在三维 (3D) 环境中, 例如在淋巴结或组织中。迄今为止, 大多数体外系统依赖于二维 (2D) 表面, 如细胞培养板或盖玻片。为了最佳地模拟体外生理条件, 我们利用一个简单的3D 胶原基质。胶原蛋白是细胞外基质 (ECM) 的主要成分之一, 被广泛用于构成3D 矩阵。对于3D 成像, 最近开发的光片显微技术 (也称为单平面光照显微镜) 具有采集速度快、穿透深度大、漂白低、photocytotoxicity 等特点。此外, 光片显微镜对长期测量特别有利。在这里, 我们描述了一个优化的协议如何建立和处理人类免疫细胞,例如, 在3D 胶原基质中的主要人细胞毒性 T 淋巴细胞 (CTL) 和自然杀伤细胞 (NK), 用于活细胞成像的光片显微镜和固定样本。给出了图像采集和细胞迁移分析的步骤。特别着重强调了取样准备和数据分析的关键步骤和因素。该协议可用于其他类型的悬浮细胞在3D 胶原基质和不限于免疫细胞。
Introduction
大多数关于迁移细胞的知识来自2D 实验1,2,3, 通常是在玻璃或塑料表面进行的一种文化/成像皿。然而, 在大多数情况下, 生理场景需要3D 微环境, 其中胞外基质 (ECM) 起决定性作用。ECM 不仅提供了维持适当细胞形态所必需的3D 结构, 而且还为许多细胞4、5的最佳功能提供了生存信号或方向性提示。因此, 需要一个3D 的环境来更好地识别细胞的功能和行为在一个更好地反映生理环境的环境中。
在人体内, 大多数细胞特别是免疫细胞, 在3D 的情况下发挥作用。例如, 活化 t 细胞巡视组织寻找靶细胞, 天真的 t 细胞通过淋巴结迁移, 寻找其同源抗原呈现细胞, 在此期间, 迁移模式和机械适应相应的胞外环境3,6,7。3D 胶原凝胶已被广泛应用为一种成熟、特征良好的3D 细胞培养系统,8、9、10。我们以前的研究表明, 主要的人类淋巴细胞是高度流动的, 以平均速度迁移, 在0.25% 的胶原基基质11中, µm/分钟大约4.8。细胞骨架的重排在移植12中起关键作用。积累的证据表明, 淋巴细胞不应用只有单一的迁移模式, 但可以切换到特定的迁移行为, 取决于位置, 微环境, 细胞因子, 趋化梯度, 和细胞外信号, 调节迁移行为以不同的方式3。
为了可靠地分析免疫细胞的功能和行为, 例如, 迁移, 突起形成或水泡运输, 能够快速可靠地获取相对较大的3D 卷中的图像是很有好处的。对于3D 成像, 最近开发的光片显微技术 (也称为单平面照明显微镜) 提供了一个满意的解决方案13,14。在成像采集过程中, 生成一个薄的静态光片来照亮样品。这样, 在焦点平面上, 可以同时照亮大面积而不影响离平面单元。此功能使高采集速度与大幅降低漂白和 photocytotoxicity。本文介绍了如何使用光片显微镜对人体免疫细胞进行可视化, 以及如何分析3D 场景中的迁移。
Protocol
Representative Results
Discussion
大多数的体外检测是在2D 表面进行的, 例如细胞培养板、培养皿或盖玻片, 而在体内细胞, 特别是免疫细胞中, 大多经历了3D 微环境。新出现的证据显示, 免疫细胞的迁移模式在2D 和3D 方案17之间有所不同。此外, 肿瘤细胞的表达谱也不同于2D 和 3 d 培养组织18,19,20。因此, 为了更好地模拟体外?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢临床 Hemostaseology 和输血医学研究所提供献血者的血液;卡门 Hässig 和科拉恩维尔·霍查为优秀的技术帮助。我们感谢 Rettig (萨尔大学) 的修改 pMAX 矢量, 罗兰 Wedlich-Söldner (Muenster 大学) 为原 LifeAct-红宝石结构, 和基督教勇克 (萨尔大学), 以产生 LifeAct-mEGFP 结构。该项目由 Sonderforschungsbereich 1027 (项目 A2 到 B.Q.) 和 894 (项目 A1 M.H.) 提供资金。光片显微镜由 DFG (广州: 256/4 19-1 FUGG) 资助。
Materials
| Fibricol, bovine collagen solution | Advanced Biomatrix | #5133-20ML | Collagen matrix |
| 0.5 M NaOH Solution | Merck | 1091381000 | for neutralizing Fibricol solution |
| Ultra-Low melting agarose | Affymetrix | 32821-10GM | Sample preparation in low c[Col] |
| Dynabeads Untouched Human CD8 T Cells Kit | Thermo Fisher | 11348D | Isolation of primary human CD8+ T cells from PBMC |
| Dynabeads Human T-Activator CD3/CD28 for T Cell Expansion and Activation | Thermo Fisher | 11132D | Activation of CTL populations |
| Human recombinant interleukin-2 | Thermo Fisher | PHC0023 | Stimulation of cultured CTL |
| P3 Primary solution kit | Lonza | V4XP-30XX | Transfection |
| α-PFN1 antibody, rabbit, IgG | Abcam | ab124904 | IF |
| Alexa Fluor 633 Phalloidin | Thermo Fisher | A22284 | IF |
| CellMask Orange Plasma membrane Stain | Thermo Fisher | C10045 | Fluorescent cell label |
| Tween 20 | Sigma | P1379-250mL | IF |
| Triton X-100 | Eurobio | 018774 | IF |
| DPBS Dulbecco's phopsphate buffered saline | Thermo Fisher | 14190250 | IF |
| Bovine serum albumin | Sigma | A9418-100G | IF |
| Goat α Rabbit 568, IgG, rabbit | Thermo Fisher | A-11011 | IF |
| Lightsheet Z.1 (Light-sheet microscopy) | Zeiss | N.A. | |
| Cell culture hood | Thermo Fisher | HeraSafe KS | |
| Cell culture incubator HERACell 150i | Thermo Fisher | N.A. | |
| Centrifuge 5418 and 5452 | Eppendorf | N.A. | |
| Pippettes | Eppendorf | 3123000039, 3123000020, 3123000063 | |
| Pippette tips | VWR | 89079-444, 89079-436, 89079-452 | |
| 15 mL tubes | Sarstedt | 62.554.002 | |
| Capillaries 50 µL | VWR (Brand) | 613-3373 | Zeiss LSFM sample preparation |
| Plunger for capillaries | VWR (Brand) | BRND701934 | "Stamps with Teflon tip" LSFM sample preparation |
| MColorPhast pH stips | Merck | 1095430001 | to test pH of neutralized Fibricol |
| BD Plastipak 1mL syringes | BD | Z230723 ALDRICH | Alternative sample preparation |
| Modeling clay (Hasbro Play-Doh A5417EU7) | Play-Doh | N.A. | |
| Imaris file converter | Bitplane | available at http://www.bitplane.com | Convert imaging files to Imaris file format |
| Imaris 8.1.2 (MeasurementPro, Track, Vantage) | Bitplane | available at http://www.bitplane.com | Analysis of 3D and 4D imaging data |
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