John Elliot (2010) CIL:7913, 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.

此数据集系由国立卫生研究院3T3成纤维细胞在聚苯乙烯上培养的非重叠区域的三份复制品之一。每个样本以孔号标识。每张图像包含该区域的四个图像作为时间序列，其中第一张图像为相衬图像，第二张图像通道为受Tenascin-C启动子驱动的EGFP报告基因向量调控的不稳定表达，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）。图像采集于蔡司Axiovert 100显微镜，使用蔡司A-plan 10x Ph1 0.25 NA物镜观察样本，并借助CoolSnapFX相机进行2 x 2合并记录。滤光片配置如下：1）用于成像DAPI、EGFP和TxRed的自定义二向色倍频器（型号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。测试培养物过夜培养后，用磷酸盐缓冲盐水（PBS）冲洗，并使用由4%（质量/体积）聚乙二醇（PEG）8000、100 mM 1,4-哌嗪二乙烷磺酸（PIPES）、10 mM 乙二醇双（2-氨基乙基）乙二酸（EGTA）组成的微管稳定缓冲液（MTSB），pH 6.9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小时于室温。移除固定剂，使用含有10 ng/mL的Tx Red C2-马来酰亚胺和2 ng/mL Dapi的0.05% Triton X100在PBS中的溶液固定2小时。移除染色剂，用PBS/3% BSA和0.05%叠氮化钠及PBS冲洗细胞，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的溶液作为防褪色试剂，以便进行成像。使用70%乙醇擦拭孔底，然后干燥擦拭后再进行成像。本数据集旨在测量单个细胞群体中EGFP荧光强度的分布。利用采集到的图像集，通过ImageJ插件执行以下图像分析任务：1）对Txred图像（细胞体通用染色）进行手动阈值分割；2）使用所得掩码在DAPI和EGFP图像上定义细胞感兴趣区域（ROI）；3）从DAPI图像中确定每个ROI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，并确定仅在该3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，可用来识别细胞簇（即具有多于1个核的细胞）、碎片或部分细胞（即无核细胞）、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的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。