John Elliot (2010) CIL:8848, 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.

本数据集系由多聚苯乙烯培养的 NIH 3T3 纤维母细胞非重叠区域构成的三份独立数据集之一。每一份数据集均以孔号标识。每张图像包含该区域的四个连续时间序列图像。首张图像为相衬图像，第二图像通道为受 Tenascin-C 启动子驱动的 destabilized EGFP 报告基因载体控制，第三图像为 Texas Red C2-maleimide（用于染色细胞体），第四图像为 Dapi（用于染色细胞核）。图像采集于 Zeiss Axiovert 100 显微镜，采用 Zeiss A-plan 10x Ph1 0.25 NA 物镜观察，并使用 CoolSnapFX 摄像头进行 2 x 2 合并拍摄。使用的滤光片如下：1. 适用于成像 DAPI、EGFP 和 TxRed 的定制二向色倍增光束分离器（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。

实验方案：细胞在传递周期中于 TCPS 培养皿中以每孔约 1000 个细胞进行接种。测试培养物在过夜培养后用 PBS 漂洗，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-maleimide 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% 碘化钠以及 PBS 漂洗细胞，然后在每个孔中加入含有 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% 乙醇擦拭孔底，然后进行干燥擦拭，随后进行成像。

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，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 图像上定义细胞感兴趣区域 (ROI)；3. 通过 DAPI 图像确定每个 ROI 中的细胞核数量，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 扩散 3 像素并确定仅在该 3 像素扩散区域内的像素强度，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，可以用于识别细胞簇（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，以解决对图像中染色和/或荧光检测的疑问。