Robust Cell Cycle Analysis Using the FlowSight Imaging Flow Cytometerwith the
Bivariate Cell Cycle Kit for G2/M Analysis
C e l l C y c l e
The Flow cytometric analysis of cell DNAcontent is widely used for the estimationof cell cycle phase distributions. Adding auorescently-labeled cell cycle phase-specicantibody to complement the DNA-binding dyetypically used for cell cycle analysis allowsfor a robust bivariate cell cycle assay that canaccurately identify mitotic cells. By furtherrunning such a bivariate analysis on an imaging ow cytometer, the mitotic population can bevisually analyzed to identify and enumeratecells in specic stages of mitosis. This studydemonstrates such an approach by pairing the Amnis
imaging ow cytometerwith the EMD Millipore FlowCellect
BivariateCell Cycle Kit for G2/M Analysis.The cell cycle can be divided into two distinctstages. The rst stage, Interphase, consists of the G1, S, and G2 phases in which cells are active,growing, and replicating DNA. Cell division takesplace in the second, M-phase or mitotic phase.
Measuring Cell Cycle
Figure 1: Typical histogram of cell cycle analysis by DNAcontent using a single uorescent DNA-binding dye.
N o r m a l i z e d F r e q u e n c y
Researchers have employed various strategies to closelyinterrogate each of the critical steps of the various phasesin the cell cycle. For example, analysis of cells in S-phaseprovides a direct measurement of newly synthesizedDNA, while the ratio of cells in the G2 and M phases isindicative of the number of cells undergoing mitosis. Inall, a comprehensive understanding of cell cycle behaviorprovides useful information which can be important ininterpreting the intrinsic nature of cell proliferation andassist in the development of anti-neoplastic agents.
Improving Cell Cycle Analysis
The FlowSight imaging ow cytometer operates like aconventional ow cytometer but provides up to 12 imagesof every cell, including brighteld, darkeld and up to nineuorescence channels. This unique combination providesimportant new capabilities, such as visual vericationof each object acquired, and measurement of the spatiallocation of uorescence on, within, or between cells.Visual verication removes the guesswork from gating andcan accelerate assay development and optimization byproviding visual conrmation of assay performance in anyindividual cell or group of cells. Automated quantitativemeasurement of uorescence localization and morphologyenables a broad range of applications that would beimpossible by traditional ow cytometry with statisticallyrobust, quantitative image analyses that are not availableby conventional microscopy.The FlowCellect Bivariate Cell Cycle Kit for G2/M Analysis(cat. no. FCCH025103) simplies the study of the cellcycle by providing a cell cycle phase-specic antibody(Anti-phospho-Histone H3 (Ser10) – Alexa Fluor 488conjugated monoclonal antibody) in addition to a DNA-binding dye (Propidium Iodide) to yield a robust bivariateanalysis in ow cytometry applications. Moreover, the kitincludes a BrdU precursor to allow for BrdU measurementby the conjugated antibody and an RNase reagent toensure that the PI intensity is directly proportional to DNAcontent only. This method of analysis reveals not only thedistribution of cells in particular phases of the cycle, butalso provides insight into the molecular and functionalmechanisms associated within the cell cycle.