Ex vivo detection of apoptotic trophoblast cells applying flow cytofluorometry and immunocytochemistry using M30 antibody directed to the cytokeratin 18 neo-epitope.
Apoptosis of placental trophoblast cells has become the subject of intensive research. Recently, a monoclonal antibody (M30) directed against a neo-epitope of cytokeratin 18, that is formed after cleavage of this cytoskeletal protein by caspases, was shown to be of advantage over other tests for the detection of trophoblast cell apoptosis. In the present study, we describe a method for the enrichment of highly pure villous trophoblast cells based on the proteolytic digestion of placental tissue, density gradient separation of dissected cells, and immunoelimination of contaminating, non-trophoblast cells employing an antibody to the HLA class I antigen. The high purity (94-99%) of the trophoblast cell preparation was shown by antibody staining for cytokeratin 7 and absence of vimentin. Furthermore, we demonstrate that after a simple permeabilization and fixation step with 90% methanol and using the M30 CytoDeath, FITC-conjugated antibody, apoptotic trophoblast cells could be distinguished from non-apoptotic cells by flow cytofluorometry in a highly quantitative and sensitive fashion. Our protocol is an improvement over previously used methods such as immunocytochemistry as it allows to differentiate rapidly between competent and apoptotic trophoblast cells by the quantitative method of flow cytofluorometry.
Krol
Janna
J
Klinische Forschergruppe der Frauenklinik der Technischen Universitaet Muenchen, Klinikum rechts der Isar, Germany.
Mengele
Karin
K
Ottl-Mantchenko
Irina
I
Welk
Anita
A
Wasilewitsch
Irina
I
von Steinburg
Stephanie Pildner
SP
Schneider
Karl-Theodor M
KT
Schmitt
Manfred
M
D000911:Antibodies, Monoclonal / Q000032:analysis*; D017209:Apoptosis; D045744:Cell Line, Tumor; D000939:Epitopes / Q000276:immunology*; D005260:Female; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D053552:Keratin-7; D007633:Keratins / Q000032:analysis / Q000276:immunology*; D011247:Pregnancy; D015203:Reproducibility of Results; D014327:Trophoblasts / Q000737:chemistry* / Q000166:cytology; D014746:Vimentin / Q000032:analysis
D000911:Antibodies, Monoclonal; D000939:Epitopes; C507497:KRT7 protein, human; D053552:Keratin-7; D014746:Vimentin; D007633:Keratins