Warawdekar Ujjwala M
Cancer Research Institute (CRI) Lab, Advanced Centre for Treatment, Research and Education in Cancer, Tata Memorial Centre, Navi Mumbai 410210, India.
Homi Bhabha National Institute, Anushakti Nagar, Mumbai 400085, India.
J Biomol Tech. 2019 Apr;30(1):1-6. doi: 10.7171/jbt.19-3001-001.
This protocol was developed to assess communication in tumor cells and to provide a dependable and standardized assay for the determination of gap junction function. The method is noninvasive; in this method, the cell population under study is divided such that 1 fraction is loaded with a lipophilic cell plasma membrane permeable dye, calcein acetoxymethyl ester, that is hydrolyzed upon cellular uptake by cytoplasmic esterases to yield calcein, a fluorescent and membrane-impermeable molecule. The other fraction is loaded with 1,1'-dioctadecyl-3,3,3',3' tetramethylindodicarbocyanine perchlorate (DiD)/1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate [Dil; DilC(3)], which is a lipophilic membrane dye that diffuses laterally to stain the entire cell membrane, is impermeable, and attains an orange-red fluorescence upon incorporation into membranes. The 2 fractions are mixed and incubated under coculture conditions. Calcein with MW 890 kD is transferred to the DiD/DiI-stained cells through gap junctions. The assessment of this uptake is made with confocal imaging and quantitated using flow cytometry. Cell lines representing cancer of the breast as well as a nontransformed cell line developed from the buccal mucosa were analyzed for gap junction competency. Confocal imaging with acquisition at specific time points during the treatment and flow cytometry gave a qualitative and quantitative analysis of the passage of molecules through the gap junctions. Here, the method has been combined to obtain images as well as quantitation and is a simple and effective approach in assessing the functional competency of gap junction in epithelial cells.
本方案旨在评估肿瘤细胞中的通讯情况，并为确定间隙连接功能提供一种可靠且标准化的检测方法。该方法是非侵入性的；在此方法中，将所研究的细胞群体进行分割，使其中一部分加载亲脂性的可透过细胞质膜的染料——钙黄绿素乙酰氧基甲酯，该染料在细胞摄取后被细胞质酯酶水解，产生钙黄绿素，这是一种荧光且不能透过细胞膜的分子。另一部分加载1,1'-二辛基-3,3,3',3'-四甲基吲哚羰花青高氯酸盐（DiD）/1,1'-二辛基-3,3,3',3'-四甲基吲哚碳菁[Dil；DilC(3)]，这是一种亲脂性膜染料，可横向扩散以染遍整个细胞膜，不可渗透，并入膜后发出橙红色荧光。将这两部分混合并在共培养条件下孵育。分子量为890 kD的钙黄绿素通过间隙连接转移至DiD/DiI染色的细胞中。通过共聚焦成像对这种摄取情况进行评估，并使用流式细胞术进行定量分析。对代表乳腺癌的细胞系以及从颊黏膜发育而来的非转化细胞系进行间隙连接能力分析。在处理过程中的特定时间点进行采集的共聚焦成像和流式细胞术对分子通过间隙连接的情况进行了定性和定量分析。在此，该方法已结合起来以获取图像以及进行定量分析，是评估上皮细胞中间隙连接功能能力的一种简单有效的方法。
