John Elliot (2010) CIL:7828, Mus musculus, permanent cell line cell. CIL. Dataset

This is part of a triplicate data set of non-overlapping fields of NIH 3T3 fibroblasts cultured on polystyrene. Each set is indicated by the well number. Each image contains 4 images of each field as a time series. The first image is the phase contrast image. The second image channel is Tenascin-C promoter driven destabilized EGFP reporter vector. The third image is Texas Red C2-maleimide (to stain cell body), and the fourth image is Dapi (to stain nuclei).

Images were collected on a Zeiss Axiovert 100. The samples were viewed with a Zeiss A-plan 10x Ph1 0.25 NA objective and recorded with a CoolSnapFX camera using 2 x 2 binning. The filters were as follows: 1.) A custom dichroic multipass beam splitter optimized for imaging DAPI, EGFP and TxRed (part# BS51019+400dclp, Chroma Technology, VT); 2.) DAPI excitation filter-360/40 nm; 3.) DAPI emission filter-460/50 nm; 4.) EGFP excitation filter-470/40 nm; 5.) EGFP emission filter-525/50 nm; 6.) TxRed excitation filter-568/24 nm; 7.) TxRed emission filter-630/60 nm. Autofocus on the TxRed color channel was performed at each location before the series of images were collected.

Protocol: The cells were seeded on TCPS dishes at ~1000 cells/well during a passage cycle. The test cultures were incubated overnight before being rinsed with PBS and then fixed with 300 uM m-maleimidobenzoyl-N-hydroxysuccinimidyl ester (MBS) in microtubule stabilizing buffer (MTSB) composed of 4% (w/v) polyethylene glycol (PEG) 8000, 100 mM 1,4-piperazinediethanesulfonic acid (PIPES),10 mM ethylene glycol-bis(2-aminoethylether)-N,N,N’,N’-tetraacetic acid (EGTA), pH 6.9 for at least 16h at RT. The fixative was removed and solution of 0.05% Triton X100 in PBS containing 10 ng/mL of Tx Red C2-maleimide and 2 ng/mL DAPI for 2h. The staining solution was removed, cells were rinsed with PBS/3% BSA, and 0.05% sodium azide and PBS. A 50% glycerol/10 mM Tris, pH 8.0 containing 2ng/mL DAPI and 0.9g/l 1,4-diazobicyclo[2,2,2]octane (DABCO) as an antifade reagent was then added to each well for imaging purposes. The bottom of the wells were wiped with 70% ethanol wipe and then a dry wipe before imaging.

Purpose: The purpose of the dataset was to measure the distribution of EGFP fluorescence intensities within individual cells in the population. Using the image sets collected, a plugin for ImageJ was used to perform the following image analysis tasks. 1.) The Txred images (which is a general purpose cell body stain) were segmented by manual thresholding. 2.) The resulting mask was used to define cell ROIs on the DAPI and EGFP images. 3.) The number of nuclei in each ROI is determined from the DAPI image and the integrated intensity of the EGFP signal in the cell is determined from ROI in the EGFP image. 4.) A local background intensity around each cell in the EGFP image is determined by dilating the ROI by 3 pixels and determining the pixel intensities in only the 3 pixel dilation area. When this data is placed in a spread sheet, you can use the results to identify cell clusters (i.e. have more than 1 nuclei), debris or partial cell (i.e. no nuclei), poor EGFP/cell measurements (i.e. high background intensity). The spreadsheet can be used to measure the distribution of EGFP cell intensities within a population of cells. The phase images were collected as quality control and validation data. The phase images provide a human validation mechanism if there are questions about the staining and/or fluorescence detection in an image.

References: 1. Langenbach, K.J., Elliott, J.T., Tona, A., and Plant, A.L. (2006) Evaluating the correlation between fibroblast morphology and promoter activity on thin films of extracellular matrix proteins. BMC-Biotechnology 6(1):14. 2.Elliott, J.T., Halter, M., Woodward, J.T., Langenbach, K.J., Tona, A., Plant, A.L. (2008) Evaluating the performance of fibrillar collagen films formed at polystyrene surfaces as cell culture substrates. Biointerphases. 3(2):19-28.

本数据集为非重叠区域中NIH 3T3纤维母细胞在聚苯乙烯培养皿上培养的三份重复数据集之一。每个集合通过孔号进行标识。每个图像包含每个区域的4张时间序列图像。首张图像为相衬图像。第二通道图像由Tenascin-C启动子驱动的EGFP报告基因矢量不稳定（编号BS51019+400dclp，Chroma Technology，VT）。第三图像为Texas Red C2-马来酰亚胺（用于染色细胞体），第四图像为Dapi（用于染色细胞核）。图像收集于Zeiss Axiovert 100显微镜。样品使用Zeiss A-plan 10x Ph1 0.25 NA物镜进行观察，并通过CoolSnapFX相机以2 x 2合并模式进行记录。滤光片配置如下：1）针对DAPI、EGFP和TxRed成像优化的定制二向色倍频器（编号BS51019+400dclp，Chroma Technology，VT）；2）DAPI激发滤光片-360/40 nm；3）DAPI发射滤光片-460/50 nm；4）EGFP激发滤光片-470/40 nm；5）EGFP发射滤光片-525/50 nm；6）TxRed激发滤光片-568/24 nm；7）TxRed发射滤光片-630/60 nm。在收集图像系列之前，在每个位置对TxRed颜色通道进行自动对焦。

实验流程：细胞在TCPS培养皿中以约1000个细胞/孔的密度在传代周期中接种。测试培养物在用PBS清洗并固定于含有4%（w/v）聚乙二醇8000、100 mM 1,4-哌嗪二乙烷磺酸（PIPES）、10 mM乙二醇双（2-氨基乙基）醚-N,N,N’，N’-四乙酸（EGTA），pH 6.9的微管稳定缓冲液（MTSB）中的300 uM m-马来酰亚胺苯甲酰-N-羟基琥珀酰亚胺酯（MBS）中至少16小时，于室温下进行孵育。移除固定剂，用含有10 ng/mL的Tx Red C2-马来酰亚胺和2 ng/mL Dapi的0.05% Triton X100在PBS中的溶液进行2小时的染色。移除染色溶液，用PBS/3% BSA和0.05%叠氮化钠以及PBS清洗细胞。然后向每个孔中加入含有2 ng/mL Dapi和0.9g/l 1,4-二氮杂双环[2,2,2]辛烷（DABCO）的50%甘油/10 mM Tris，pH 8.0溶液，作为防褪色试剂，以便进行成像。使用70%乙醇擦拭孔底，然后进行干燥擦拭，随后进行成像。

目的：本数据集的目的是测量单个细胞群体中EGFP荧光强度的分布。利用收集到的图像集，通过ImageJ插件执行以下图像分析任务：1）对Txred图像（通用细胞体染色）进行手动阈值分割；2）使用所得掩码在DAPI和EGFP图像上定义细胞区域；3）通过DAPI图像确定每个区域中的核数量，并从EGFP图像的ROI中确定细胞中EGFP信号的积分强度；4）通过将ROI膨胀3像素并确定仅3像素膨胀区域内的像素强度，确定EGFP图像中每个细胞周围的局部背景强度。当将这些数据放入电子表格中时，可以利用结果来识别细胞簇（即具有多个核）、碎片或部分细胞（即无核）、EGFP/细胞测量不良（即高背景强度）。电子表格可用于测量细胞群体中EGFP细胞强度的分布。相衬图像作为质量控制和验证数据收集。相衬图像提供了一种人工验证机制，以解决对图像中染色和/或荧光检测的疑问。

参考文献：1. Langenbach, K.J.，Elliott, J.T.，Tona, A.，Plant, A.L. (2006) 评估成纤维细胞形态与细胞外基质蛋白薄膜上启动子活性的相关性。BMC-Biotechnology 6(1):14。2. Elliott, J.T.，Halter, M.，Woodward, J.T.，Langenbach, K.J.，Tona, A.，Plant, A.L. (2008) 评估在聚苯乙烯表面形成的纤维状胶原蛋白薄膜作为细胞培养基底的性能。Biointerphases. 3(2):19-28。