John Elliot (2010) CIL:7865, 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成纤维细胞在聚苯乙烯上培养的非重叠区域的一部分，形成了一套三份复制品。每个样本以孔号进行标识。每个图像包含该区域的四幅时序图像。首幅图像为相位对比图像，次之则为由Tenascin-C启动子驱动的EGFP报告基因载体不稳定表达，第三幅图像采用Texas Red C2-马来酰亚胺（用于细胞体染色），第四幅图像则使用Dapi（用于染色细胞核）。图像采集于Zeiss Axiovert 100显微镜，使用Zeiss A-plan 10x Ph1 0.25 NA物镜观察样本，并通过CoolSnapFX相机进行记录，采用2 x 2的合并方式。使用的滤波器包括：1）定制二向色倍频束分离器，优化用于DAPI、EGFP和TxRed成像（编号BS51019+400dclp，Chroma Technology，VT）；2）DAPI激发滤波器-360/40 nm；3）DAPI发射滤波器-460/50 nm；4）EGFP激发滤波器-470/40 nm；5）EGFP发射滤波器-525/50 nm；6）TxRed激发滤波器-568/24 nm；7）TxRed发射滤波器-630/60 nm。在采集图像系列之前，对TxRed颜色通道进行了自动对焦。实验方案：在传代周期中，将细胞以约1000个细胞/孔的密度接种于TCPS培养皿中。测试培养物过夜培养后，用PBS清洗，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，至少在室温下固定16小时。移除固定剂，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，用PBS/3% BSA和0.05%叠氮化钠及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溶液作为抗褪色剂，以备成像。用70%乙醇擦拭孔底，然后干燥擦拭，再进行成像。目的：该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）手动阈值分割Txred图像（作为通用细胞体染色）；2）使用所得掩码在DAPI和EGFP图像上定义细胞区域；3）从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个像素并确定3个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。将此数据放置在电子表格中，可以使用结果来识别细胞簇（即具有超过1个核的细胞）、碎片或部分细胞（即无核），以及EGFP/细胞测量不佳（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。相位图像作为质量控制和数据验证采集。相位图像为人眼验证提供了机制，如果对图像中的染色和/或荧光检测有疑问。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.