Cell proliferation and apoptosis in the primary enamel knot measured by flow cytometry of laser microdissected samples.
Laser capture microdissection (LCM) uniquely allows the selection of specific cell populations from histological sections. These selected cells are then catapulted into a test tube without any contamination from surrounding tissues. During the last ten years, many significant results have been achieved, particularly at the level of DNA and RNA where amplification techniques are available. However, where amplification procedures are difficult, the benefits of LCM diminish. To overcome such difficulties, a novel approach, combining laser capture microdissection and flow cytometry, has been tested here for detection of apoptosis and proliferation in tissue bound cell populations without any amplification steps. The mouse cap stage molar tooth germ was used as a model. At the centre of the inner enamel epithelium, the primary enamel knot is a clearly defined apoptotic population with minimal proliferation, flanked by the highly proliferative cervical loops on each side. Thus within the tooth germ epithelium at this stage, two distinct populations of cells are found side by side. These populations were selected by laser capture microdissection and then analysed by flow cytometry for apoptosis and proliferation. Flow cytometric results correlated well with immunohistochemical findings, demonstrating the success and sensitivity of this combined procedure.
Matalova
E
E
Department of Physiology and Pathophysiology, University of Veterinary and Pharmaceutical Sciences, Brno, Czech Republic. matalova@iach.cz
Dubska
L
L
Fleischmannova
J
J
Chlastakova
I
I
Janeckova
E
E
Tucker
A S
AS
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D002452:Cell Count; D049109:Cell Proliferation; D015925:Cryopreservation; D004658:Enamel Organ / Q000166:cytology*; D004847:Epithelial Cells / Q000166:cytology; D005434:Flow Cytometry; D005865:Gestational Age; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D053685:Laser Therapy / Q000379:methods*; D051379:Mice; D042282:Microdissection / Q000379:methods*; D008963:Molar / Q000196:embryology; D018809:Proliferating Cell Nuclear Antigen / Q000032:analysis; D012680:Sensitivity and Specificity; D019237:Tooth Cervix / Q000166:cytology / Q000196:embryology; D014083:Tooth Germ / Q000166:cytology
D018809:Proliferating Cell Nuclear Antigen