自动定量分析细胞计数程序使用ImageJ插件
Summary
This paper describes the quantification of hemocytometer and migration/invasion micrographs through two new open-source ImageJ plugins Cell Concentration Calculator and migration assay Counter. Furthermore, it describes image acquisition and calibration protocols as well as discusses in detail the input requirements of the plugins.
Abstract
健康的ImageJ的国家研究所是一个功能强大，免费的图像处理软件套件。 ImageJ的具有可有效地用于计算各种生物颗粒全面粒子分析算法。当计数大量细胞样品，血细胞计数仪呈现与问候时间的瓶颈。同样地，从与ImageJ的插件细胞计数迁移/侵袭测定法计数的膜，虽然准确，分外劳动密集型的，主观的和声名狼藉用于使手腕疼痛。为了满足这一需求，我们开发了内ImageJ的两个插件用于自动血球（或已知体积）和迁移/侵袭细胞计数的唯一任务。这两个插件依靠获得高品质的显微照片以最小的背景的能力。他们很容易使用，对于大样本容量，内置分析工具的快速计算和分析，帮助计数的校准优化。通过组合的核心原则与自动计数算法和后计数分析细胞计数的S，这大大增加了与该迁移测定可以不准确性的任何损失进行处理的容易性。
Introduction
体外细胞计数是在广泛的组织培养实验的一个重要的基本技术。准确地确定在培养细胞的数目为实验的重复性和标准化1,2-必不可少的。细胞计数可以手动使用血细胞计数器以及使用各种自动化方法，各有各的优点和缺点3,4,5来执行。大多数的细胞计数的自动化方法属于两类，那些使用库尔特原理或流式细胞仪中的一个。犁刀计数器利用细胞电阻，以确定细胞数量和大小。他们是快速，准确，价格比流式细胞仪。然而，它们很少用于仅细胞计数，由于其相当大的成本相比，人工计数3。流式细胞仪，在另一方面，是昂贵的，但它们有许多应用，例如细胞计数，细胞形状，ST的分析ructure和测量内部细胞标记物4,5。使用以下两种原则的机器都可以从许多制造商。人工计数是可以承受的，但费时，并受偏压而自动化方法来提供关于整个人工计数所需要的时间的一小部分，但使用昂贵的机器6。
其他常见的细胞培养过程是在体外细胞运动性测定法，即，细胞迁移和侵入7。迁移和侵袭测定法通常用于研究细胞运动和侵袭响应于趋化反应。另外，它们被广泛地用于研究胚胎发育，分化，炎症反应，并转移多种细胞类型7-11的。已迁移或侵入通过迁移测定的多孔膜细胞可以在两种不同的方式来量化。首先，通过用荧光染料染色的细胞中，分离来回中号使用荧光读取器12的膜，和定量。定量的这种方法的一个限制是，没有记录可以保持膜的，并且没有可能用于进一步分析13。第二量化方法被用于迁移/侵袭细胞中固定并用荧光染料或更常见的是，与细胞学染料如结晶紫，甲苯胺蓝染料或苏木精;然后细胞用这些膜这是一个非常耗时的任务12,13的倒置显微图像手动定量。
为了克服手动细胞计数的缺点，二可靠和准确的对细胞浓度和迁移测定自动细胞计数器被开发。这些自动细胞计数算法是为ImageJ的开发与使用Oracle的Java计算机语言的插件。 ImageJ的是HEA国立研究所开发一个公共的，广泛使用的图像处理工具第l（NIH）14,15;因此，写这些插件ImageJ的有利于方便地集成到生物群落。
相比手工计数细胞计数的自动化，确保高吞吐量和再现性。尽管其它可用的软件和插件可用于通过图像分析5,16,17，细胞浓度计算器插件来计算细胞浓度是快，也可以处理的细胞和治疗的稀释液。此外，这两个计数器所有结果和计算可以保存和出口。在本文所描述的两个插件是通过使用解剖显微镜的使用相衬显微镜的活细胞成像和大视场（全膜捕捉）成像迁移实验膜进行了优化。该插件是免费提供从下载安装说明：http://peng.lab.yorku.ca/imagej-plugins。
Protocol
Representative Results
Discussion
关键步骤，故障排除和限制
自动计算方法的本质，特别是那些颗粒的分析，就必须确定这些粒子的数学能力。因此，这两种细胞浓度计算器和迁移测定计数器的精度majorly依赖图像保真度，也就是，所捕获的图像如何酷似细胞样品或迁移测定膜。因此的至上重要性跟随显微镜和相关软件校准协议尽可能最好。这包括限制的背景噪声，减少了不必要的颗粒，捕捉明亮，均匀对焦的?…
Declarações
The authors have nothing to disclose.
Acknowledgements
This work was supported by the Canadian Institute of Health Research to CP (OR 142730 and OR 89931). We would like to thank Jelena Brkic for her initial idea of binary particle analysis in ImageJ.
Materials
| HyClone Classical Liquid Media: RPMI 1640 – With L-Glutamine |
Fisher Scientific | SH3002702 | Cell culturing media |
| Fetal bovian serum (FBS) | GIBCO BRL | P00015 | Media suppliment |
| HTR8/SVneo trophoblast cell line | Cells were obtained from Dr. Charles Graham (Queen’s University, Kingston, Canada) | Software is designed to work with any cell line. | |
| Trypsin | GIBCO BRL | 27250-018 | Prepared as 0.20% (w/v) in 10uM EDTA 1X PBS |
| Accutase | Innovative Cell Technologies | AT104 | |
| 10 cm cell culture plates | SARSTEDT | 833902 | Any tissue culture treated plates will be suitable |
| Transwell Polyester Membrane Inserts – 8.0µm Pore size | Costar 3422 ordered from Fisher Scientific | 7200150 | For 24-well plates; Pore size: 8.0µm; 6.5mm diameter; 0.33cm2 growth area |
| HARLECO Hematology Stains and Reagents, EMD Millipore – Soluntions 1, 2 & 3 | EMD Millipore and ordered from VWR | 65044A, B, & C | Hemacolor stain set consists of three 500mL (16.9oz.) poly bottles & includes a methanol fixative (Solution 1), an eosin or acid stain (Solution 2), and a methylene blue or basic stain (Solution 3) |
| Cotton Tipped Applicator | Puritan Medical | 806-WC | |
| Single-edge industrial razor blades | VWR | 55411 – 055 | Thickness: 0.30 mm (0.012") |
| Microscope Slides – Precleaned/Plain | Fisher Scientific | 12550A3 | Dimentions: 25 X 75 X 1.0 mm |
| Fisherbrand Cover Glasses – Rectangles no. 1 | Fisher Scientific | 12-545E | Thickness: 0.13 to 0.17mm; Size: 50 x 22mm |
| Fisher Chemical Permount Mounting Medium | Fisher Scientific | SP15-500 | |
| Leica Stereo dissecting microscope | Leica Microsystems | The microsope is equipped with Leica microscope camera Model MC170 HD & camera software is Leica App. Suite (LAS E2) Version 3.1.1 [Build: 490]. Microscope parts: LED3000 Spot Light Illumination Model: MEB126, Leica M80 Optic Carrier Model M80, Objective achromat 1.0X, WD=90mm Model: MOB306 & Objective achromat 0.32X, WD=303mm Model: MOB315, Video Objective 0.5X Model: MTU-293, | |
| Hemacytometer | Assistant Germany | 0.100 mm Depth – 0.0025 mm2 | |
| Olympus inverted light microscope | Olympus Corporation | CKX41SF | The microsope is equipped with Lumenera Infinity 1-2 2.0 Megapixel CMOS Color Camera & camera software is Infinity analyze Version 6.5.2 |
| Laminar flow cabinet 1300 Series A2 | Thermo Scientific | Model: 1375 | Any laminar flow cabinet for cell culture work will be suitable |
| Cell culture incubator | Thermo Scientific | Model: 370 | Any cell culture incubator will be suitable – Cells were cultured under humidefied environment, 5% CO2, 37 °C |
| ImageJ | NIH | Version 1.50e | Minimum version required |
| Java Runtime Environment | Oracle | Version 1.8.0_66 | Minimum version required |
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