小鼠皮肤角质细胞的分离和染色,用于细胞周期特定分析细胞蛋白表达的量度细胞测定
Summary
该协议描述了如何从小鼠模型中分离皮肤角蛋白细胞,用金属标记的抗体染色,以及通过质量细胞测定法分析染色细胞,以便分析不同细胞周期阶段感兴趣的蛋白质的表达模式。
Abstract
该协议的目标是使用从小鼠皮肤表皮分离的单细胞,以细胞周期相关的方式检测和量化蛋白质表达变化。有七个重要步骤:表皮从真皮分离,表皮消化,用顺铂染色表皮细胞群,样品条形码,用金属标记的抗体染色细胞周期标记和蛋白质兴趣,通过质量细胞学检测金属标记抗体,以及分析不同细胞周期阶段的表达。与组织学方法不同,这种方法的优点是有可能在细胞周期的不同阶段对单个细胞中>40个不同标记的表达模式进行测定。这种方法还允许对蛋白质表达进行多变量相关性分析,这种分析比组织学/成像方法更具可量化性。该协议的缺点是需要暂停单个细胞,从而导致组织部分染色所提供的位置信息丢失。这种方法可能还需要加入额外的标记,以识别粗细胞悬浮液中的不同细胞类型。在超塑性皮肤病模型分析中,该方案的应用十分明显。此外,通过添加系骨特异性抗体,可调整该方案,用于分析特定亚型细胞(如干细胞)。该协议也可以用于分析其他实验物种的皮肤细胞。
Introduction
在分析癌症等高塑性疾病的动物模型时,基因表达与细胞周期阶段的相关性仍然是一个挑战。这一挑战的一部分是共同检测具有增殖标记的感兴趣的蛋白质(POI)。增殖细胞可在各种细胞周期阶段找到,包括G1、S、G2和M.Ki67是最常用的增殖标记之一,在细胞周期的所有阶段表达。它已被广泛用于分析人类和小鼠组织1,2,3。然而,与其他一般增殖标记一样,Ki67不识别单个细胞周期阶段。更精确的方法将胸腺素核苷类类似物(如溴氧尿酸(BrdU)结合到正在积极复制其基因组的细胞中(即S相)4、5。使用核苷酸类似物的一个缺点是需要在分析前几小时管理它们以活的动物。使用抗体在固定组织部分通常检测到 Ki67 和 BrdU。这种方法的一个优点是,POI的位置可以在组织架构(例如,皮肤表皮的基础层)中确定。这种方法也不需要组织分离,可能导致基因表达的变化。一个缺点是组织固定或组织处理OCT冷冻或石蜡切片可能遮挡抗体靶点(即抗原)。抗原的回收通常需要热或组织消化。染色强度的量化也可能具有挑战性。这是由于染色、截面厚度、信号检测和实验偏置的变化。此外,在大多数典型的实验室设置中,可以同时检测到数量有限的标记。然而,较新的多路复用染色方法有望克服这些限制;例如成像质量细胞测定和提拉姆德信号放大6,7。
流式细胞测定是另一种检测增殖细胞的强大技术。它允许对同一细胞中的标记进行多倍检测,但大多数非造血细胞类型需要组织分离。增殖细胞的分析通常通过使用结合DNA的染料(例如,碘化钠(PI))8。流式细胞学还允许更精确地测定细胞周期阶段,当与BrdU结合9的检测相结合时。BrdU/PI 流式细胞测量虽然是一种强大的方法,但确实存在其缺点。如果不纳入相位特异性抗体,它无法解决G2/M和G0/G1相。然而,可以使用的抗体数量受到细胞自荧光、荧光辐射的光谱溢出和使用补偿控制的限制。这种限制标志着与POIs共同检测细胞周期标记的表达更具挑战性和艰巨性。一个较简单的方法是使用质量细胞测定10,11。该技术使用具有较窄检测光谱的金属结合抗体。一旦细胞被金属标记的抗体染色,它们就会蒸发,并且细胞测量时间(CyTOF)质谱仪检测到的金属。由于这些特性,质量细胞仪能够使用现有平台10、11对>40个不同标记进行多路检测。此外,还可以用金属对样品进行条形码,从而节省宝贵的抗体,同时减少样品到样品的染色变异性。另一方面,质量细胞学确实有一些缺点。对于非血液衍生细胞,市售的金属标记抗体数量有限。与使用荧光DNA染料相比,DNA含量的定量不太敏感,与荧光流细胞测定相比,质量细胞测量具有较低的信号检测动态范围。
此处描述的方案旨在分析从小鼠皮肤中新分离的角蛋白细胞 (KCs) 中的细胞周期动力学,并使用质量细胞测定来描述这些细胞中的细胞周期特定蛋白表达。该协议也可以与培养的细胞一起使用,或适应其他细胞类型。
Protocol
Representative Results
Discussion
本文概述的协议可在8小时左右完成。最终结果是悬浮在KCs中富集的细胞,这些细胞可以以细胞周期依赖的方式分析蛋白质表达。以前的几项研究已经概述了从人类和小鼠皮肤分离的IC的方法16,25。这些研究还包括用于流式细胞测定26的SC分离方案。然而,以前没有详细描述过将IC分离与使用质量细胞测定分析蛋白质表达和细胞周期动力学相?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项工作的支持来自皮肤病系、科罗拉多大学盖茨再生医学中心和科罗拉多大学皮肤病中心形态和表型核心(NIAMS P30 AR057212)。作者感谢 UC 癌症中心流式细胞测量共享资源和支持赠款 (NCI P30 CA046934) 用于大规模细胞仪的运行,并感谢核心的 Karen Helm 和 Christine Childs 在流量和质量细胞测量方面的专家建议技术。
Materials
| 12-well plate | Cell Treat | 229512 | |
| Intercalator solution | Fluidigm | 201192A | 125 µM – Ir intercalator solution |
| Paraformaldehyde | Electron Microscopy Sciences | 30525-89-4 | 16 % PFA |
| Strainer cap flow tubes | Fisher/Corning | 352235 | 35 µm pore size |
| Cell sieve | Fisher | 22363547 | 40 μm pore size |
| Cell detatchment solution | CELLnTEC | CnT-ACCUTASE-100 | Accutase |
| Iodine Solution | ThermoFisher/Purdue | 67618-151-17 | Betadine 7.5%-iodine surgical scrub |
| Barcode permeabilization buffer | Fluidigm | 201060 | Cell-ID 20-Plex Pd Barcoding Kit |
| Barcodes | Fluidigm | 201060 | Cell-ID 20-Plex Pd Barcoding Kit |
| pH strips | EMD | 9590 | colorpHast |
| DMEM | Hyclone | SH30022.01 | Dulbecco’s Modified Eagle Media |
| Fine forceps | Dumont & Fils | 0109-5-PO | Dumostar #5 |
| Curved precision forceps | Dumont & Fils | 0109-7-PO | Dumostar #7 |
| Calibration Beads | Fluidigm | 201078 | EQ Four Element Calibration Beads |
| HBSS | Gibco | 14175-095 | Hank's Balanced Salt Solution |
| Water | Fisher | SH30538.03 | Hyclone Molecular Biology grade water |
| Iododeoxyuridine | Sigma | I7125 | IdU |
| Cryo vials | ThermoFisher | 366656PK | internal thread |
| Cell Staining buffer | Fluidigm | 201068 | Maxpar Cell Staining buffer |
| Fix & Perm buffer | Fluidigm | 201067 | Maxpar Fix & Perm buffer |
| Fix I buffer | Fluidigm | 201065 | Maxpar Fix I buffer |
| Phosphate buffered saline | Rockland | MB-008 | Metal free 10x PBS |
| isopropanol-freezing container | ThermoFisher | 5100-0001 | Mr.Frosty |
| Sodium hydroxide | Fisher | BP359-500 | NaOH |
| Petri dish | Kord-Valmark | 2900 | Supplied by Genesee 32-107 |
| 15 mL conical | Olympus/Genesee | 28-101 | |
| 50 mL conical | Olympus/Genesee | 28-106 | |
| 6-well plate | Cell Treat | 229506 | |
| Cisplatin | Sigma | 479306 | |
| Dispase II | Sigma/Roche | 4942078001 | |
| DMSO | Sigma | D2650 | |
| FBS | Atlanta Biologicals | S11150 | |
| Hydrochloric acid | Fisher | A144-212 | |
| Nuclear Antigen Staining permeabilization buffer | Fluidigm | 201063 | |
| Nuclear Antigen Staining buffer | Fluidigm | 201063 | |
| Trypan blue | Sigma | T8154 | |
| Tuberculin syringe | BD | 309626 | |
| Type IV Collagenase | Worthington Bioscience | CLSS-4 |
References
- Guzinska-Ustymowicz, K., Pryczynicz, A., Kemona, A., Czyzewska, J. Correlation between proliferation markers: PCNA, Ki-67, MCM-2 and antiapoptotic protein Bcl-2 in colorectal cancer. Anticancer Research. 29 (8), 3049-3052 (2009).
- Ladstein, R. G., Bachmann, I. M., Straume, O., Akslen, L. A. Ki-67 expression is superior to mitotic count and novel proliferation markers PHH3, MCM4 and mitosin as a prognostic factor in thick cutaneous melanoma. BioMedCentral Cancer. 10, 140 (2010).
- Ryan, W. K. Activation of S6 signaling is associated with cell survival and multinucleation in hyperplastic skin after epidermal loss of AURORA-A Kinase. Cell Death & Differentiation. , (2018).
- Magaud, J. P. Double immunocytochemical labeling of cell and tissue samples with monoclonal anti-bromodeoxyuridine. Journal of Histochemistry & Cytochemistry. 37 (10), 1517-1527 (1989).
- Dolbeare, F., Gratzner, H., Pallavicini, M. G., Gray, J. W. Flow cytometric measurement of total DNA content and incorporated bromodeoxyuridine. Proc Natl Acad Sci U S A. 80 (18), 5573-5577 (1983).
- Toth, Z. E., Mezey, E. Simultaneous visualization of multiple antigens with tyramide signal amplification using antibodies from the same species. Journal of Histochemistry & Cytochemistry. 55 (6), 545-554 (2007).
- Stack, E. C., Wang, C., Roman, K. A., Hoyt, C. C. Multiplexed immunohistochemistry, imaging, and quantitation: a review, with an assessment of Tyramide signal amplification, multispectral imaging and multiplex analysis. Methods. 70 (1), 46-58 (2014).
- Kraemer, P. M., Petersen, D. F., Van Dilla, M. A. DNA constancy in heteroploidy and the stem line theory of tumors. Science. 174 (4010), 714-717 (1971).
- Dolbeare, F., Gratzner, H., Pallavicini, M. G., Gray, J. W. Flow cytometric measurement of total DNA content and incorporated bromodeoxyuridine. Proceedings of the National Academy of Sciences U S A. 80 (18), 5573-5577 (1983).
- Bandura, D. R. Mass cytometry: technique for real time single cell multitarget immunoassay based on inductively coupled plasma time-of-flight mass spectrometry. Analytical Chemistry. 81 (16), 6813-6822 (2009).
- Bjornson, Z. B., Nolan, G. P., Fantl, W. J. Single-cell mass cytometry for analysis of immune system functional states. Current Opinion in Immunology. 25 (4), 484-494 (2013).
- Behbehani, G. K., Bendall, S. C., Clutter, M. R., Fantl, W. J., Nolan, G. P. Single-cell mass cytometry adapted to measurements of the cell cycle. Cytometry A. 81 (7), 552-566 (2012).
- Brodie, T. M., Tosevski, V. High-Dimensional Single-Cell Analysis with Mass Cytometry. Current Protocols in Immunology. 118, 5.11.11-15.11.25 (2017).
- McCarthy, R. L., Duncan, A. D., Barton, M. C. Sample Preparation for Mass Cytometry Analysis. Journal of Visual Experimentation. 122, (2017).
- Lichti, U., Anders, J., Yuspa, S. H. Isolation and short-term culture of primary keratinocytes, hair follicle populations and dermal cells from newborn mice and keratinocytes from adult mice for in vitro analysis and for grafting to immunodeficient mice. Nature Protocols. 3 (5), 799-810 (2008).
- Zhang, L. Defects in Stratum Corneum Desquamation Are the Predominant Effect of Impaired ABCA12 Function in a Novel Mouse Model of Harlequin Ichthyosis. PLoS One. 11 (8), e0161465 (2016).
- Zunder, E. R. Palladium-based mass tag cell barcoding with a doublet-filtering scheme and single-cell deconvolution algorithm. Nature Protocols. 10 (2), 316-333 (2015).
- Sumatoh, H. R., Teng, K. W., Cheng, Y., Newell, E. W. Optimization of mass cytometry sample cryopreservation after staining. Cytometry A. 91 (1), 48-61 (2017).
- Finck, R. Normalization of mass cytometry data with bead standards. Cytometry A. 83 (5), 483-494 (2013).
- Trowbridge, I. S., Thomas, M. L. CD45: an emerging role as a protein tyrosine phosphatase required for lymphocyte activation and development. Annual Review of Immunology. 12, 85-116 (1994).
- Torchia, E. C., Boyd, K., Rehg, J. E., Qu, C., Baker, S. J. EWS/FLI-1 induces rapid onset of myeloid/erythroid leukemia in mice. Molecular and Cellular Biology. 27 (22), 7918-7934 (2007).
- Liu, Z. A Simplified and Efficient Method to Isolate Primary Human Keratinocytes from Adult Skin Tissue. Journal of Visual Experimentation. (138), (2018).
- Jensen, K. B., Driskell, R. R., Watt, F. M. Assaying proliferation and differentiation capacity of stem cells using disaggregated adult mouse epidermis. Nature Protocols. 5 (5), 898-911 (2010).
- Germain, L. Improvement of human keratinocyte isolation and culture using thermolysin. Burns. 19 (2), 99-104 (1993).
- Fluhr, J. W. Impact of anatomical location on barrier recovery, surface pH and stratum corneum hydration after acute barrier disruption. British Journal of Dermatology. 146 (5), 770-776 (2002).
- Webb, A., Li, A., Kaur, P. Location and phenotype of human adult keratinocyte stem cells of the skin. Differentiation. 72 (8), 387-395 (2004).
- Torchia, E. C., et al. A genetic variant of Aurora kinase A promotes genomic instability leading to highly malignant skin tumors. Recherche en cancérologie. 69 (18), 7207-7215 (2009).
- Frei, A. P. Highly multiplexed simultaneous detection of RNAs and proteins in single cells. Nature Methods. 13 (3), 269-275 (2016).
