John Elliot (2010) CIL:8963, 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.

本数据集系由美国国立卫生研究院3T3成纤维细胞在聚苯乙烯上培养而成的非重叠区域数据集的三份复制品之一。每个样本以孔号进行标识。每个图像包含该区域四个时间序列图像。首张图像为相衬图像。第二通道图像由Tenascin-C启动子驱动的非稳定EGFP报告基因质粒构成。第三图像为Texas Red C2-马来酰亚胺（用于染色细胞质），第四图像为Dapi（用于染色细胞核）。图像采集于蔡司Axiovert 100显微镜。样本采用蔡司A-plan 10x Ph1 0.25 NA物镜观察，并利用CoolSnapFX相机通过2 x 2合并方式记录。使用的滤光片如下：1.) 适用于成像DAPI、EGFP和TxRed的自定义二向色多通道光束分离器（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。在采集图像序列之前，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/孔的密度接种于传代周期中。测试培养物在过夜培养后用磷酸盐缓冲盐水（PBS）冲洗，然后用微管稳定缓冲液（MTSB）中300 uM m-马来酰亚胺苯甲酰-N-羟基琥珀酰亚胺酯（MBS）固定，MTSB由4%（质量/体积）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）组成，pH 6.9，在室温下至少固定16小时。去除固定剂，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，细胞用PBS/3% BSA和0.05%叠氮化钠及PBS冲洗。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）作为抗褪色剂的50%甘油/10 mM Tris，pH 8.0溶液，以便进行成像。使用70%乙醇擦拭培养皿底部，然后进行干燥擦拭，以进行成像。目的：本数据集旨在测量单个细胞在细胞群体中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（这是一种通用细胞质染色）；2.) 使用得到的掩码在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中的细胞核数量，并从EGFP图像中的ROI确定细胞内EGFP信号的积分强度；4.) 通过将ROI膨胀3个像素，并确定仅在该3个像素膨胀区域内像素的强度，确定EGFP图像中每个细胞周围的局部背景强度。将此数据放入电子表格中，可以使用结果来识别细胞簇（即具有超过1个核），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。相衬图像作为质量控制和数据验证收集。相衬图像提供了一种人工验证机制，以解决图像中染色和/或荧光检测的问题。
参考文献：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.