title: An imaging flow cytometric method for measuring cell division history and molecular symmetry during mitosis.
abstract: Asymmetric cell division is an important mechanism for generating cellular diversity, however, techniques for measuring the distribution of fate-regulating molecules during mitosis have been hampered by a lack of objectivity, quantitation, and statistical robustness. Here we describe a novel imaging flow cytometric approach that is able to report a cells proliferative history and cell cycle position using dye dilution, pH3, and PI staining to then measure the spatial distribution of fluorescent signals during mitosis using CCD-derived imagery. Using Jurkat cells, resolution of the fluorescently labeled populations was comparable to traditional PMT based cytometers thus eliminating the need to sort cells with specific division histories for microscopy. Subdividing mitotic stages by morphology allowed us to determine the time spent in each cell cycle phase using mathematical modeling approaches. Furthermore high sample throughput allowed us to collect statistically relevant numbers of cells without the need to use blocking agents that artificially enrich for mitotic events. The fluorescent imagery was used to measure PKCζ protein and EEA-1+ endosome distribution during different mitotic phases in Jurkat cells. While telophase cells represented the favorable population for measuring asymmetry, asynchronously dividing cells spent approximately 43 seconds in this stage, explaining why they were present at such low frequencies. This necessitated the acquisition of large cell numbers. Interestingly we found that PKCζ was inherited asymmetrically in 2.5% of all telophasic events whereas endosome inheritance was significantly more symmetrical. Furthermore, molecular polarity at early mitotic phases was a poor indicator of asymmetry during telophase highlighting that, though rare, telophasic events represented the best candidates for asymmetry studies. In summary, this technique combines the spatial information afforded by fluorescence microscopy with the statistical wealth and objectivity of traditional flow cytometry, overcoming the key limitations of existing approaches for studying asymmetry during mitosis.
authors.affiliation: Flow Cytometry Laboratory, London Research Institute, Cancer Research UK. Andy.Filby@cancer.org.uk
authors.forename: Andrew
authors.initials: A
authors.lastname: Filby
authors.forename: Esperanza
authors.initials: E
authors.lastname: Perucha
authors.forename: Huw
authors.initials: H
authors.lastname: Summers
authors.forename: Paul
authors.initials: P
authors.lastname: Rees
authors.forename: Prabhjoat
authors.initials: P
authors.lastname: Chana
authors.forename: Susanne
authors.initials: S
authors.lastname: Heck
authors.forename: Graham M
authors.initials: GM
authors.lastname: Lord
authors.forename: Derek
authors.initials: D
authors.lastname: Davies
mesh_terms: D000818:Animals; D002455:Cell Division / Q000502:physiology*; D005434:Flow Cytometry / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D019044:Image Cytometry / Q000295:instrumentation / Q000379:methods*; D019169:Jurkat Cells / Q000166:cytology; D008856:Microscopy, Fluorescence / Q000379:methods; D008938:Mitosis / Q000502:physiology*; D011493:Protein Kinase C / Q000378:metabolism; D000099221:Protein Kinase C zeta
chemical_list: D011493:Protein Kinase C; D000099221:Protein Kinase C zeta