一种用于小数目造血干细胞的西方印迹协议
1Division of Experimental Hematology,Cincinnati Children’s Hospital Medical Center, 2Department of Cell and Developmental Biology, Abramson Family Cancer Research Institute,Perelman School of Medicine, University of Pennsylvania, 3Department of Obstetrics and Gynecology, Southwest Hospital,Third Military Medical University
Summary
通过对500只造血干细胞或祖细胞进行分析, 优化了标准的西方印迹协议。优化包括仔细处理细胞样本, 限制管道之间的传输, 并直接株溶藻 Laemmli 样本缓冲区中的细胞。
Abstract
造血干细胞 (HSCs) 是罕见的细胞, 与小鼠骨髓中仅含有2.5万表型的长期再生 HSCs。对 HSCs (500-1.5万细胞) 的小数目进行了优化, 并适合于对其进行分析。表型 HSCs 纯化, 准确计数, 直接裂解 Laemmli 样品缓冲。用月桂酸钠聚丙烯酰胺凝胶电泳 (SDS 页) 分析了含有等量细胞的裂解物, 并根据标准的西方印迹协议制备并处理了该印迹。使用此协议, 可以对 2000-5000 HSCs 进行例行分析, 在某些情况下, 数据可以从很少500个单元中获得, 而大多数出版物中报告的2万到4万个单元格。本议定书应普遍适用于其他造血细胞, 并能通过标准实验室程序对少量细胞进行常规分析。
Introduction
造血干细胞 (HSCs) 是自我更新的细胞, 可以引起所有的血血统。它们是骨髓中相对稀少的细胞, 使得生物化学分析变得困难。适合于分析稀有细胞的方法, 如流式细胞术, 对于定量测定细胞表面标记物和胞内蛋白的相对数量非常有用。然而, 细胞内蛋白的分析需要使用细胞通透程序来实现抗体的存取, 而不是所有细胞表面的表位都能存活这些程序1,2。此外, 在不同的蛋白质亚型或裂解产物之间鉴别的抗体通常不能用于流式细胞术, 因此调查人员仍然依靠西方的印迹进行某些类型的分析。
裂解物细胞的印迹分析是大多数实验室的常规程序。细胞可以在保存细胞表面分子表位的本地条件下纯化, 细胞裂解物随后可以被制备和分析。然而, 用西方印迹对稀有的原代细胞种群中的蛋白质进行分析, 可以要求 euthanizing 大量的动物获得足够的细胞。通过对几个步骤进行小的调整, 传统的西方印迹协议能够检测出相对较小数量的 HSCs (500-1.5万, 取决于感兴趣的蛋白质) 的蛋白质。这些调整包括准确计数细胞, 仔细处理细胞颗粒, 减少细胞间的转移, 以最小化细胞损失, 并株溶藻一个定义数量的细胞与集中加载缓冲包含蛋白酶体和磷酸酶抑制剂。许多发表的报告包括西部污点获得与2万或更多 HSCs3,4,5,6,7;这个简单的程序将减少在4到40倍之间产生等效数据所需的细胞和实验动物的数量。该协议旨在以每个单元为基础对结果进行规范化, 而不是内部控制。这使得检测蛋白质水平的全面降低, 如果数据被规范化到内部控制, 可以忽略。本文描述了在细胞基础上规范化的重要性, 用于分析基因表达数据8, 同样的原理也适用于用西方印迹来定量蛋白质。此优化的协议对于需要分析少量单元格的任何人都是有用的。
Protocol
所有程序必须按照机构动物使用和护理指南执行。该程序是为分析小鼠造血干细胞和祖细胞 (HSCs 和高压), 但可以适应其他细胞群体的分析而制定的。 1. 流式细胞仪分离小鼠 HSCs 和高压的研究 收获小鼠骨髓细胞如描述在文献6。注: 在图 1和图 2所示的数据中, 骨髓是从 C57BL/6J 小鼠身上采集的。 按?…
Representative Results
500-2000 纯化 HSCs 和高压钠灯的代表性结果如图 1和图 2所示。图 1中的β肌动蛋白信号可以从从一只老鼠的骨髓中纯化出来的 500 HSCs 和高压钠灯中检测出来。请注意, 将裂解物装入1.5 毫米井, 产生了比加载到3.0 毫米井更强的500高压信号。图 2是 EIF4G 的西方印迹和 Rps6 (p-Rps6) 的磷酸化, ?…
Discussion
西方印迹是一种常见的技术检测特定的蛋白质和激活信号通路的组织或细胞。通过对通常使用的程序进行小的调整, 我们能够例行地检测15种不同的蛋白质 (材料表), 在 1.5万 HSCs, 在某些情况下, 在 500 HSCs。该协议中最关键的步骤是: 1) 精确计数单元格, 2) 最小化管之间的传输数, 3) 株溶藻单元格直接与 Laemmli 样本缓冲区。当株溶藻的细胞颗粒与 Laemmli 样品缓冲, 我们发现, 而不是删除所有…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
美国国立卫生研究院资助 R01 CA149976 支持这项工作。
Materials
| sodium dodecyl sulfate (SDS) | Fisher Scientific | BP166-500 | SDS-PAGE |
| TEMED | Fisher Scientific | BP150-20 | SDS-PAGE |
| 30% Acrylamidel Bis solution | Bio-Rad | 1610158 | SDS-PAGE |
| 1.5M Tris-HCl pH8.8 | TEKNOVA | T1588 | SDS-PAGE |
| 1.0M Tris-HCl pH6.8 | TEKNOVA | T1068 | SDS-PAGE |
| Ammonium Persulfate | Fisher Scientific | BP179-25 | SDS-PAGE |
| mini-protean 3 comb | Bio-Rad | 1653360 | SDS-PAGE |
Mini-PROTEAN Tetra Cell |
Bio-Rad | 1658005EDU | SDS-PAGE |
| Transfer System | Bio-Rad | 1704155EDU | transfer |
| PowerPac HC Power Supply | Bio-Rad | 1645052EDU | electrophoresis |
| Imaging System | Bio-Rad | 1708195 | signal detection |
| 4x Laemmli Sample Buffer | Bio-Rad | 1610747EDU | sample preparation |
| 10x Tris/Glycine/SDS Electrophoresis Buffer | Bio-Rad | 1610732EDU | electrophoresis |
| 2-Mercaptoethanol | Bio-Rad | 1610710EDU | sample preparation |
| RTA Mini PVDF Transfer Kit, for 40 blots | Bio-Rad | 1704272 | transfer |
| Protease Inhibitor Cocktail | Sigma | 11697498001 | sample preparation |
| Phosphatase Inhibitor Cocktailrs | Gold Biotechnology | GB-450-1 | sample preparation |
| EIF4G | Cell signaling | 2469 | WB antibody, validated in 1000 cells antibody concentration: 1:1000 reference(figure): Fig2 |
| p-Rps6 | Cell signaling | 4858 | WB antibody,validated in 1000 cells antibody concentration: 1:1000 reference(figure): Fig2 |
| actin(HRP conjugated) | abcam | ab49900 | WB antibody,validated in 500 cells antibody concentration: 1:5000 reference(figure): Fig1, Fig2 |
| LC-3 | Abcam | ab48394 | WB antibody,validated in 15000 cells antibody concentration: 1:1000 reference(figure): ref5 (Fig5) |
| S6 | Cell Signaling | 2317 | WB antibody,validated in 15000 cells antibody concentration: 1:1000 reference(figure): ref5 (Fig4) |
| 4EBP1 | Cell Signaling | 9644 | WB antibody, validated in 15000 cells antibody concentration: 1:1000 reference(figure): ref5 (Fig4) |
| p-4EBP1 | Cell Signaling | 2855 | WB antibody,validated in 15000 cells antibody concentration: 1:1000 reference(figure): ref5 (Fig4) |
| TSC2 | Cell Signaling | 4308 | WB antibody,validated in 15000 cells antibody concentration: 1:1000 reference(figure): ref5 (Fig4) |
| HSP90 | Cell Signaling | 4877 | WB antibody,validated in 15000 cells antibody concentration: 1:1000 reference(figure): ref5 (Fig5) |
| PTEN | Cell Signaling | 9188 | WB antibody,validated in 15000 cells antibody concentration: 1:1000 reference(figure): ref5 (FigS1) |
| mTOR | Cell Signaling | 2983 | WB antibody,validated in 15000 cells antibody concentration: 1:1000 reference(figure): ref5 (FigS1) |
| p-mTOR | Cell Signaling | 5536 | WB antibody,validated in 15000 cells antibody concentration: 1:1000 reference(figure): ref5 (FigS1) |
| PP2AB | Cell Signaling | 4953 | WB antibody,validated in 15000 cells antibody concentration: 1:1000 reference(figure): ref5 (FigS1) |
| p-AMPKa | Cell Signaling | 2535 | WB antibody,validated in 15000 cells antibody concentration: 1:1000 reference(figure): ref5 (FigS1) |
| actin | Santa Cruz | sc-47778 | WB antibody,validated in 15000 cells antibody concentration: 1:3000 reference(figure): ref5 (Fig4) |
| lineage depletion kit (mouse) | Miltenyi Biotec | 130-090-858 | hematopoietic stem and progenitor cells purification |
| LS columns | Miltenyi Biotec | 130-041-305 | hematopoietic stem and progenitor cells purification |
| SCF | PeproTech | 250-03 | phosphorylation stimulation |
| APC-Cy7 c-kit antibody | ThermoFisher | A15423 | cell sorting |
| anti-B220 | ThermoFisher | 48-0452-82 | cell sorting |
| anti-CD3e | ThermoFisher | 48-0031-82 | cell sorting |
| anti-Mac1 | ThermoFisher | 48-0112-82 | cell sorting |
| anti-Gr1 | ThermoFisher | 48-5931-82 | cell sorting |
| anti-Ter119 | ThermoFisher | 48-5921-82 | cell sorting |
| Spacer Plates with 1.0 mm Integrated Spacers | Bio-Rad | 1653311 | SDS-PAGE |
| BSA | Research Products International | A30075 | membrane blocking |
| serum | Atlanta Biologicals | A12450 | medium supplement |
| cell counter | Invitrogen | AMQAF1000 | cell counting |
| hemocytometer | ThermoFisher | S17040 | cell counting |
| flim | Denville Scientific | E3012 | signal detection |
| medical film processor | Konica | SRC-101A | signal detection |
| Supersignal West Femto Maximum Sensitivity Substrate | Therom Scientific | 34096 | signal detection |
| Allegra X-15R Centrifuge (Rotor SX4750A) | Beckman Coulter | X-15R | sample preparation |
| BLUEstain protein ladder | Gold Biotechnology | P007-500 | electrophoresis |
| cell sorter | BD | FACSAria II | sample preparation |
| Incublock | Denville Scientific | I0510 | electrophoresis |
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Citazione di questo articolo
Cai, X., Zheng, Y., Speck, N. A. A Western Blotting Protocol for Small Numbers of Hematopoietic Stem Cells. J. Vis. Exp. (138), e56855, doi:10.3791/56855 (2018).