John Elliot (2010) CIL:7889, 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 NIH 3T3 纤维母细胞在聚苯乙烯上培养的非重叠区域的三份复制品之一。每个集合通过孔号进行标识。每张图像包含每个区域的时间序列的4张图像。第一张图像为相衬图像。第二通道图像由 Tenascin-C 启动子驱动的去稳定 EGFP 报告基因向量产生。第三张图像为 Texas Red C2-马来酰亚胺（用于染色细胞体），第四张图像为 Dapi（用于染色细胞核）。图像采集于 Zeiss Axiovert 100 显微镜。样本使用 Zeiss A-plan 10x Ph1 0.25 NA 物镜观察，并利用 CoolSnapFX 摄像头以 2 x 2 集束方式记录。过滤器配置如下：1.) 专为成像 DAPI、EGFP 和 TxRed 优化的定制二色性倍频束分裂器（部件号 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。在采集图像序列之前，在每个位置上对 TxRed 颜色通道进行自动对焦。

实验流程：在传代周期中，将细胞接种于 TCPS 培养皿中，每孔约 1000 个细胞。测试培养物过夜培养后，用 PBS 洗涤，然后用含有 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) 固定至少 16 小时。移除固定剂，然后用含有 10 ng/mL Tx Red C2-马来酰亚胺和 2 ng/mL Dapi 的 0.05% Triton X100 在 PBS 中的溶液固定 2 小时。移除染色溶液，用 PBS/3% BSA 和 0.05% 硝酸钠以及 PBS 洗涤细胞。随后在每个孔中加入含有 2ng/mL Dapi 和 0.9g/l 1,4-二氮杂双环[2,2,2]辛烷 (DABCO) 作为防褪色剂的 50% 甘油/10 mM Tris，pH 8.0 溶液，以用于成像。用 70% 乙醇擦拭孔底，然后进行干燥擦拭以进行成像。

研究目的：本数据集旨在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，使用 ImageJ 插件执行以下图像分析任务：1.) 通过手动阈值分割 Txred 图像（这是一项通用的细胞体染色）；2.) 使用所得掩模在 DAPI 和 EGFP 图像上定义细胞感兴趣区域（ROI）；3.) 从 DAPI 图像中确定每个 ROI 中的核数，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 扩散 3 像素并确定仅 3 像素扩散区域的像素强度，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，可以使用结果来识别细胞簇（即具有超过 1 个核的细胞）、碎片或部分细胞（即无核），以及 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。相衬图像的收集作为质量控制和数据验证。

相衬图像提供了一种人类验证机制，以防对图像中的染色和/或荧光检测存在疑问。

参考文献：1. Langenbach, K.J., Elliott, J.T., Tona, A., and Plant, A.L. (2006) 评估成纤维细胞形态与细胞外基质蛋白薄膜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质的性能。Biointerphases. 3(2):19-28。