A three-colour flow cytometry technique for measuring trophoblast intracellular antigens: The relative expression of TAP1 in human cytotrophoblast and decidual cells

Flow cytometry is conventionally used to measure cell-surface antigen expre ssion. However, many antigens are found within the cytoplasm, and it is nec essary to fix and permeabilize cells to enable antibodies to gain access to them. In this study we have established the conditions for studying intrac ellular antigens in human trophoblast cells by flow cytometry using an anti body to TAP1 (a key molecule in the process of Class I MHC assembly). We ha ve previously shown by immunocytochemistry that TAP1 expression is apparent ly greater on Class I positive extravillous cytotrophoblast than on any oth er fetal or maternal tissue. However, as immunohistochemistry is not quanti tative we have used three-colour flow. cytometry to measure the expression of TAP1 in different trophoblast populations. Villous and extravillous cyto trophoblast were identified in first trimester and term placental and decid ual digests on the basis of their expression of cytokeratin and Class I MHC antigens. The level of expression of TAP1 for each population was investig ated using a commercial kit that determines the number of antibody-binding sites per cell. TAP expression was found to be three- to fivefold higher in extravillous cytotrophoblast, confirming our previous findings. The techni ques developed here are directly applicable to the measurement of other int racellular molecules in trophoblast, in particular cytokines. (C) 2000 Harc ourt Publishers Ltd.