Al-Nasiry S, Geusens N, Hanssens M, Luyten C, Pijnenborg R
Department of Obstetrics and Gynaecology, University Hospital Gasthuisberg, Katholieke Universiteit Leuven, Leuven, Belgium.
Hum Reprod. 2007 May;22(5):1304-9. doi: 10.1093/humrep/dem011. Epub 2007 Feb 16.
The current techniques for quantifying trophoblast viability, migration and invasion are mainly limited by the need to sacrifice the cells during the test procedure. In this study, the vital dye AB (AB) was used to quantify cell number and viability of BeWo and JEG-3 choriocarcinoma cells, as well as their migration and invasion through fibronectin-coated filters.
AB was directly added to culture medium of incubated test and control cells. At various time intervals, the redox reaction, in which AB is reduced by the cells, was measured by absorbance readings at 540 and 630 nm. For cell migration and invasion, cells were cultured onto uncoated or fibronectin-coated inserts, respectively. AB reduction of migrated cells was normalized to that of control cells to calculate percentages of migration. This model was also tested in the presence of a reported inhibitor, transforming growth factor (TGF) beta.
The curve of %AB reduction versus cell number was linear, with intra- and inter-assay Coefficient of Variations of 1.88%and 2.94%, respectively. AB reduction increased with both seeding concentrations and incubation time with AB. TGFbeta treatment caused a modest decrease in AB reduction in both JEG-3 and BeWo cells. TGFbeta treatment also decreased migration in BeWo, but not in JEG-3, cells.
AB assay is a simple and reliable method for quantifying trophoblast viability, migration and invasion.
目前用于量化滋养层细胞活力、迁移和侵袭的技术主要受限于在测试过程中需要牺牲细胞。在本研究中，活性染料AB用于量化BeWo和JEG-3绒毛膜癌细胞的细胞数量和活力，以及它们通过纤连蛋白包被的滤膜的迁移和侵袭能力。
将AB直接添加到培养的测试细胞和对照细胞的培养基中。在不同时间间隔，通过在540和630nm处的吸光度读数测量AB被细胞还原的氧化还原反应。对于细胞迁移和侵袭，将细胞分别培养在未包被或纤连蛋白包被的小室上。将迁移细胞的AB还原量相对于对照细胞进行标准化，以计算迁移百分比。该模型也在存在报道的抑制剂转化生长因子（TGF）β的情况下进行了测试。
%AB还原量与细胞数量的曲线呈线性，测定内和测定间变异系数分别为1.88%和2.94%。AB还原量随接种浓度和与AB的孵育时间增加而增加。TGFβ处理导致JEG-3和BeWo细胞中AB还原量适度降低。TGFβ处理也降低了BeWo细胞的迁移，但未降低JEG-3细胞的迁移。
AB测定是一种用于量化滋养层细胞活力、迁移和侵袭的简单可靠方法。
