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 PKCf protein and EEA-11 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 PKCf 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. ' 2011International Society for Advancement of Cytometry Key termsimaging flow cytometry; cell cycle analysis; asymmetric cell division; flow cytometry; image analysis DETERMINING how fate decisions are made at the molecular level is key to understanding how single cell progenitors can generate different specialized cell types. This diversification can occur in two ways. When a cell divides by mitosis, equal distribution of fate determining molecules across the plane of cytokinesis will produce two identical daughter cells that can differentiate in response to extrinsic stimuli. Alternatively, if fate determinants are apportioned asymmetrically prior to cytokinesis, two qualitatively and quantitatively distinct progeny will be generated by division (1,2). Asymmetric division is a key process for driving cellular diversity in developmental biology (3-7), stem cell biology (4,(8)(9)(10) and also recently in...

Regulation of asymmetric cell division and polarity by Scribble is not required for humoral immunity

“…F5 CD8 þ T cells were prepared from lymph node preps of F5.Rag1KO mice and activated with endogenous antigen-presenting cells where specified. T cells were labelled with 2.5 mM of CellTrace Violet (CTV, Invitrogen) as described previously 32 . Cells were seeded into culture at 1 million cells 1 ml À 1 per well in 24-well plate then activated with different combinations of NP68 (10 nM, Mimotopes), anti-CD3 (145-2C11, eBioscience, either plate-bound at 10 mg ml À 1 or in solution at 0.5 mg ml À 1 ), anti-CD28 (37.51, eBioscience, plate-bound at 10 mg/ml) and recombinant ICAM1-Fc fusion (R&D Systems, plate-bound at 10 mg ml À 1 ).…”

“…S5). Cells were stained as for CFC but then labelled with propidium iodide (1 mg ml À 1 ) and acquired on a fully calibrated ImageStream X (ISx, Amnis, USA) system as described previously using  40 magnification 32,46 ). IFC analysis was performed using IDEAS software (Amnis) and Image J (v1.43, NIH, USA) as described previously 25,32 .…”

“…Cells were stained as for CFC but then labelled with propidium iodide (1 mg ml À 1 ) and acquired on a fully calibrated ImageStream X (ISx, Amnis, USA) system as described previously using  40 magnification 32,46 ). IFC analysis was performed using IDEAS software (Amnis) and Image J (v1.43, NIH, USA) as described previously 25,32 . Briefly, IDEAS was used to identify live mitotic cells with a known divisional history based on CTV signal dilution, bright-field and nuclear imagery, and up to nine objective pixel intensity-based features 32 .…”

“…In light of the fact we saw no discernible impact on the response of Scribbledeficient mice to influenza infection, we decided to assess the extent of ACD in wild-type influenza responsive transgenic CD8 þ T cells. To rapidly assess statistically relevant numbers we used an imaging flow cytometry-based (IFC) approach that has been validated for assessing ACD in lymphocytes 25,32 . This technique provides conventional flow cytometry (CFC) data with multispectral bright-field and fluorescent images, enabling assessment of ACD of all cells undergoing mitosis, and exclusion of doublet cells through strict non-biased selection, in combination with analysis of division history.…”

Monitoring cell proliferation in vitro with different cellular fluorescent dyes

“…The CFSE cytometric cell prolife- www.fhc.viamedica.pl ration test is based on the observation that, after every division, the cellular fluorescence decreases by half. Other labelling dyes: CellTrace Violet (405/450 nm) [4] and CellVue Claret (655/675 nm) are also commonly used to monitor proliferation [5]. CFSE and CellTrace Violet easily diffuse into cells, where they are cleaved by intracellular esterases to yield highly reactive, fluorescent compounds.…”