Detection of mitotic cells.
In preparation for cell division, nuclear chromatin undergoes a vital rearrangement required for the organization of chromosomes and their allocation to daughter cells. This process is initiated during G(2) phase with the most remarkable morphological manifestation being chromatin condensation. This unit provides protocols for identification and quantification of mitotic cells based on immunocytochemical detection of histone H3 phosphorylated on Ser 10 (H3-P), the critical event occurring during the G(2) to M transition (essential for chromatin condensation), using anti-H3-P, a commercially available antibody to which apoptotic cells are not reactive, concurrently with differential staining of cellular DNA. Additionally an adaptation of this method used to stain cells mounted on microscope slides for analysis by multiparameter laser scanning cytometry is also presented.
Juan
Gloria
G
Memorial Sloan-Kettering Cancer Center, New York, New York, USA.
Darzynkiewicz
Zbigniew
Z
D000818:Animals; D000906:Antibodies; D035843:Biomedical Research / Q000379:methods*; D002452:Cell Count / Q000379:methods*; D003584:Cytological Techniques / Q000379:methods; D005434:Flow Cytometry / Q000379:methods; D006657:Histones / Q000032:analysis / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D007399:Interphase; D008938:Mitosis; D010766:Phosphorylation
D000906:Antibodies; D006657:Histones