Comparison of several techniques for the detection of apoptotic astrocytes in vitro.
Implication of apoptosis in numerous physiological and pathological processes has resulted in the development of numerous methods to detect apoptosis, but none of them is adapted to all cell types. In this study, we induced apoptosis on murine immortalized astrocytes with urine from multiple sclerosis (MS) patients. Among techniques allowing the detection of apoptotic cells, only a few are adapted to adherent cells such as astrocytes. We compared several techniques (propidium iodide labelling and flow cytometry analysis, TUNEL and annexin V labelling in immunofluorescence, DNA ladder, ELISA tests to detect nucleosomes) in order to choose the method best adapted to our adherent cellular model and to discuss their practicability for the detection of apoptosis on adherent cells. For technical course, propidium iodide labelling followed by flow cytometry analysis as a quantitative technique, and TUNEL in IF (easier and quicker than propidium iodide) as a semiquantitative test were both retained as best adapted to our case. Moreover, in our model, we have observed that phosphatydilserine externalization and DNA fragmentation were concomittant after induction of apoptosis. Techniques studied in this article would allow an enlarged study of the apoptotic mechanism in several pathologies by culture of adherent cells sensitive to apoptosis in vitro.
Micoud
F
F
Unité Mixte 103 CNRS-bioMérieux, Ecole Normale Supérieure de Lyon, Lyon, France.
Mandrand
B
B
Malcus-Vocanson
C
C
D000818:Animals; D017209:Apoptosis; D001253:Astrocytes / Q000166:cytology / Q000502:physiology*; D002448:Cell Adhesion; D002460:Cell Line; D003584:Cytological Techniques; D004249:DNA Damage; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D006801:Humans; D020287:In Situ Nick-End Labeling; D051379:Mice; D009103:Multiple Sclerosis / Q000652:urine