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Chapter 16 : Flow Cytometry

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Abstract:

This chapter covers the following subjects: (i) a brief history of flow cytometry; (ii) a definition of flow cytometry; (iii) a description of the mechanics of a flow cytometer; (iv) the use of flow cytometry in detecting and quantifying human cytomegalovirus (hCMV) infected tissue culture cells; (v) the use of flow cytometry for studying apoptosis of virus-infected cells; (vi) the use of flow cytometry for measuring the effect of virus infection on the cell cycle; and (vii) the use of flow cytometry in drug susceptibility testing. Some flow cytometers are cell sorters that have the capacity to physically separate cells out of a population and collect that specific cell population. However, most of the experiments described in the chapter only require an instrument with a single argon ion laser that has the capacity to simultaneously analyze the light scatter properties of cells and two or three cell-associated fluorochromes. The chapter concentrates on a few studies that have used flow cytometry to measure in vitro drug susceptibility testing of antiviral compounds for cells infected with hCMV, human immunodeficiency virus (HIV), human herpesvirus 6 (HHV-6) and HHV8, Epstein-Barr virus (EBV), and influenza A and B viruses. In summary, the flow cytometry drug susceptibility assay for hCMV clinical isolates is accurate, rapid, and quantitative and can be automated. Expanded use of fluorochrome-labeled monoclonal antibodies to viral antigens and flow cytometry for detection and quantification of virus-infected tissue culture cells will save time and effort and make the diagnostic laboratory more efficient and productive.

Citation: McSharry J. 2009. Flow Cytometry, p 185-200. In Specter S, Hodinka R, Young S, Wiedbrauk D (ed), Clinical Virology Manual, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815974.ch16
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Image of FIGURE 1
FIGURE 1

Diagram of a flow cell with attached optical systems. As the cells pass through the flow cell (the small black square box in the center left), a laser beam intersects the stream of cells and scatters light. FW-SC passes through the forward light scatter lens, and the energy is collected by the forward light scatter photodiode. RT-SC passes through the objective lens, the beam splitter, the laser line filter, and the diffuser, and the energy is collected by the right angle scatter photomultiplier tube (PMT). If the cells are labeled with fluorescent molecules, the laser will excite these molecules, which emit light of higher energies. The emitted energies of different wavelengths pass through the objective lens and various filters and are collected and amplified by the various PMTs. The amplified signals are converted into digital information and stored in a computer for further analysis. Numbers 1 through 9 refer to the following: 1, beam splitter; 2, laser line filter, 396- to 496-nm band pass; 3, diffuser; 4, dichroic mirror 1, 570-nm long pass; 5, laser cut filter, 490-nm short cut; 6, green filter, 515- to 530-nm band pass; 7, dichroic mirror 2, 610-nm long pass; 8, orange filter, 565- to 592-nm band pass; 9, red filter, 660-nm long pass. (From McSharry, 1994, with permission.)

Citation: McSharry J. 2009. Flow Cytometry, p 185-200. In Specter S, Hodinka R, Young S, Wiedbrauk D (ed), Clinical Virology Manual, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815974.ch16
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Image of FIGURE 2
FIGURE 2

Flow cytometric analysis of uninfected and hCMV-infected HFF treated with FITC-labeled monoclonal antibody to the hCMV IE antigens. Uninfected (A) and hCMV-infected (B) HFF were permeabilized with methanol, treated with an FITC-labeled monoclonal antibody to the hCMV IE antigens, and analyzed for the percentage of antigen-positive cells by flow cytometry. The left-hand panels represent the forward and right angle light scatter analysis of the cells. Cells of the correct size to be intact cells are gated. The left-hand panels represent the analysis of fluorescence intensity of uninfected and hCMV-infected cells.

Citation: McSharry J. 2009. Flow Cytometry, p 185-200. In Specter S, Hodinka R, Young S, Wiedbrauk D (ed), Clinical Virology Manual, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815974.ch16
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Image of FIGURE 3
FIGURE 3

Effect of foscarnet on hCMV IE antigen synthesis in cells infected with a foscarnet-susceptible hCMV clinical sample. Medium containing 0 (A) or 200 (B) μM foscarnet was added to HFF monolayers. Virus-infected cells were added to the flask at an MOI of 0.01. After incubation at 37°C for 144 h, the cells were harvested with trypsin-EDTA, permeabilized with methanol, treated with a monoclonal antibody to the hCMV IE antigens, and analyzed by flow cytometry for the percentage of antigen-positive cells. Initially, the cells were analyzed for forward angle light scatter (FW-SC) versus right angle light scatter (RT-SC) to identify intact cells and exclude debris. The intact cells were gated and analyzed for the percentage of antigen-positive cells in the population. In the absence of foscarnet, 34% of the cells synthesized the hCMV IE antigens. In the presence of 200 μM foscarnet, only 17% of the cells expressed the hCMV IE antigens.

Citation: McSharry J. 2009. Flow Cytometry, p 185-200. In Specter S, Hodinka R, Young S, Wiedbrauk D (ed), Clinical Virology Manual, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815974.ch16
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Image of FIGURE 4
FIGURE 4

Effect of foscarnet on hCMV late antigen synthesis in cells infected with a foscarnet-susceptible hCMV clinical isolate. Same as in Fig. 3, except that a monoclonal antibody to the hCMV late antigen was used to identify the antigen-positive cells. (A) In the absence of foscarnet, 24.9% of the cells were positive for the late antigen. (B) In the presence of 200 μM foscarnet, only 11.7% of the cells were positive for the late antigen.

Citation: McSharry J. 2009. Flow Cytometry, p 185-200. In Specter S, Hodinka R, Young S, Wiedbrauk D (ed), Clinical Virology Manual, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815974.ch16
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Image of FIGURE 5
FIGURE 5

Effect of foscarnet on the synthesis of IE antigen in cells infected with a foscarnet-susceptible hCMV clinical isolate (A) or a foscarnet-resistant hCMV clinical isolate (B). Infection was performed as described in the legend to Fig. 3. Of the cells infected with the foscarnet-susceptible clinical isolate, 28.9% synthesize the IE antigen in the absence of foscarnet, whereas only 7.2% of the cells synthesize the IE antigen in the presence of 800 μM foscarnet. Of the cells infected with the foscarnet-resistant clinical isolate, 95.0% synthesize the IE antigen in the absence of foscarnet, whereas 72.3% of the cells infected with the resistant isolate synthesize the IE antigen in the presence of 800 μM foscarnet.

Citation: McSharry J. 2009. Flow Cytometry, p 185-200. In Specter S, Hodinka R, Young S, Wiedbrauk D (ed), Clinical Virology Manual, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815974.ch16
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Image of FIGURE 6
FIGURE 6

FACS analysis of uninfected and HIV-infected R3/X4/R5 cells expressing GFP. The left-hand side of the figure represents FACS analysis of uninfected R3/X4/R5 cell monolayers, and the right-hand side of the figure represents FACS analysis of R3/X4/R5 cell monolayers infected with cell-free HIVIIIB. After 48 h of incubation at 37°C under an atmosphere of 5% CO2, the cells were removed with trypsin-EDTA, fixed in 1% paraformaldehyde, and examined for GFP by FACS. Events with light scatter properties characteristic of intact cells were gated and analyzed for fluorescence intensity (FITC-GR-FL) versus forward angle light scatter (FW-SC). A gate was set on the uninfected cell population such that less than 1% of the fluorescent events were above the gate. Using this gate, 71.3% of the events were above the gate in the HIVIIIB -infected cells.

Citation: McSharry J. 2009. Flow Cytometry, p 185-200. In Specter S, Hodinka R, Young S, Wiedbrauk D (ed), Clinical Virology Manual, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815974.ch16
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Image of FIGURE 7
FIGURE 7

Cell-associated HIV drug susceptibility assay. R3/X4/R5 cell monolayers were infected with H9 cells chronically infected with HIVIIIB in the absence (left-hand panels) and presence (right-hand panels) of 0.05 μM AZT. After two days of incubation, the cells were analyzed for the percentage of cells expressing high levels of fluorescence intensity by FACS. The HIVIIIB -infected cells, which do not express GFP, were gated out and the R3/X4/R5 cells expressing low and high levels of fluorescence intensity were included in the gate for analysis of fluorescence intensity. To determine the percentage of HIV-infected R3/X4/R5 cells, a gate was drawn between the cell population with low fluorescence intensity and the cell population with high fluorescence intensity. In the absence of AZT, 71.4% of the R3/X4/R5 cells expressed GFP, whereas in the presence of 0.05 μM AZT, only 35.5% of the R3/X4/R5 cells expressed GFP.

Citation: McSharry J. 2009. Flow Cytometry, p 185-200. In Specter S, Hodinka R, Young S, Wiedbrauk D (ed), Clinical Virology Manual, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815974.ch16
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