Department of Pharmacology and Toxicology, Michigan State University, East Lansing, MI, USA.
Department of Pathology, College of Medicine, University of Illinois at Chicago, Chicago, IL, USA.
Toxicol Mech Methods. 2021 Jun;31(5):393-399. doi: 10.1080/15376516.2021.1904072. Epub 2021 Mar 30.
Gap junction intercellular communication (GJIC) is a necessary process for placental development. GJIC can be assessed with a parachute assay, where fluorescent dye-loaded donor cells are 'parachuted' onto acceptor cells and dye diffuses to adjacent cells with active GJIC. During co-culture, donor cells can attach, but the assay does not allow their distinction from acceptor cells, which presents as a major limitation. We have developed a modified parachute assay that permits distinction between donor and acceptor cells, using the extravillous trophoblast cell line HTR-8/SVneo and a lentiviral transduction technique. Using PKA activator CW008 as a positive control and 12-o-tetradecanoylphorbol-13-acetate as a negative control, this modified parachute assay reliably detects both enhanced and attenuated GJIC. Importantly, the ease and accuracy of quantification over currently available methods makes this modified assay optimal for automation and represents a useful tool for placental toxicological testing.
缝隙连接细胞间通讯 (GJIC) 是胎盘发育所必需的过程。GJIC 可以通过降落伞测定法进行评估，即将负载荧光染料的供体细胞“降落伞”到接受体细胞上，并且具有活性 GJIC 的染料扩散到相邻细胞。在共培养期间，供体细胞可以附着，但该测定法不允许将其与接受体细胞区分开来，这是一个主要的局限性。我们已经开发了一种改良的降落伞测定法，该方法使用绒毛外滋养层细胞系 HTR-8/SVneo 和慢病毒转导技术，可以区分供体细胞和接受体细胞。使用 PKA 激活剂 CW008 作为阳性对照，12-o-十四烷酰佛波醇-13-乙酸酯作为阴性对照，该改良的降落伞测定法可靠地检测到增强和减弱的 GJIC。重要的是，与目前可用的方法相比，该改良测定法在定量方面的简便性和准确性使其成为自动化的理想选择，并且是胎盘毒理学测试的有用工具。
