Flow cytometry phase analysis

Flow cytometry (FCM) is widely used for exploring mechanism of action of compounds that compromise proliferation since it is rapid, accurate and usable for any cellular context [5], In this chapter we want to point out technical and strategic aspects of use of FCM for cell cycle studies of a putative anticancer agent. As an example we used Edotecarin, a topi inhibitor, firstly evaluating proliferation outcome and classical DNA content analysis by propidium iodide, and then since the compound treatment produced cell cycle perturbation difficult to interprete, a two-parametric analysis by 5-bromo-deoxyuridine (BrdU) was applied for separating cell cycle phases. Moreover we put our efforts into identifing specific cell cycle arrest not easily demonstrable by previously described methods, through the use of in vitro kinetics ( pulse and chase ). Finally, in vivo assessment of efficacy and biomarkers modulation after treatment was analyzed. 

The analysis of cell-cycle progression was one of the earliest applications of flow cytometry (for review, see Darzynkiewicz et al., 2004). In this assay, fluorescence signals from cells stained with DNA-binding fluorochromes are plotted as DNA content histograms that may be analyzed by using histogram deconvolution software to quantify cell-cycle phase distributions (Rabinovitch 1994). Fluorochromes that are useful for this purpose are the plasma membrane-impermeant DNA stains, propidium iodide (PI),... 

M. Cell cycle analysis in flow cytometry Use of BrdU labelling and side scatter for the detection of the different 32. cell cycle phases. Cytotechnology 1991, 5 (Suppl. 1),... 

Fluorescence-based microbial detection systems, including epifluorescence microscopy (EFM), flow cytometry (FC) and solid-phase cytometry (SPC), lend themselves to rapid, in-situ analysis of individual microorganisms, without the need for multiplication (Lemarchand et al. 2001 Lisle et al. 2004). This review will focus on the basic principles of SPC, its advantages, disadvantages and applications, and outline some future perspectives. 

The cytostatic activities of the cholestane glycosides partly resulted from the induction of apoptosis. The apoptosis is caused by the activation of calcium dependent endonuclease. An analysis of flow cytometry pointed out that glycosides stopped the cell cycle of HL-60 at S2/M phase and induced apoptosis at GO/Gl phase . 

Laakso et al. (2001) developed a method for boron determination that uses inductively coupled plasma atomic emission spectrometry and protein removal with trichloroacetic acid before analysis. This method is feasible, accurate, and one of the fastest for boron determination during BNCT and enables a more reliable estimation of the irradiation dose. Yoshida et al. (2002) used flow cytometry to sort the cells by phases, and the boron concentration in each fraction was measured with inductively coupled plasma atomic emission spectroscopy. Obtained results revealed that sodium borocaptate and boronophenylalanine were associated with different rates of boron uptake in different phases. 

Keij, J. F. BeU-Prince, C. Steinkamp, J. A. Simultaneous analysis of relative DNA and glutathione content in viable cells by phase-resolved flow cytometry. Cytometry 1999,35,48-54. 

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