Accumulating, and then mastering varied technologies and instruments devours time as well as valuable funding resources. Often in research, a technique may be needed in only a certain experimental phase leading to an accumulation of infrequently used instruments cluttering valuable bench top space. The ideal circumstance would be to have an instrument, which could perform a variety of multi-parametric applications.
Flow cytometers have been used for an expanding set of cell analysis applications over the past 40 years. New-generation, compact flow cytometers, such as the Accuri C6 Flow Cytometer System (Fig. 1), have simplified the flow cytometric process and offer several advantages over fluorescence microscopy, microplate reading, qPCR and Western blots by enabling multi-parametric, individual-cell analysis. Pre-optimised detectors calibrated to operate within their linear range can be used to analyse a wide variety of samples, ranging from dim, barely-fluorescent, micron-sized platelets through large, >30 micron, highly-fluorescent cell lines. By incorporating a linear dynamic range greater than 6 decades, an Accuri C6 can quantitatively capture the entire scope of biological variations in a single run without the need for data acquisition optimisation or tuning. Single to hundreds of thousands of individual cells, from heterogeneous populations, in hundreds of samples, can be analysed and rare events flagged. Another powerful feature is the ability to quantitatively measure the concentration of cells or particles in samples.
GFP Transfection Studies
Flow cytometry can accurately quantitate reporter gene expression (such as, Green Fluorescent Protein, GFP) in each cell in a population being transfected. Co-transfection of a reporter plasmid and a reference plasmid can be quite variable in normal human cells, making interpretation difficult in reporter gene assays. However, using multi-parametric flow cytometry, reporter and reference plasmid expression can both be quantitated at the single cell level through the use of fluorochrome-conjugated antibodies to the transfected gene product. In addition propidium iodide can be used to monitor the DNA content of cells of varying viabilities, identifying apoptotic cells with sub G1 DNA content.
Continuous Measurement of Intracellular Ca2+
An alteration in intracellular calcium ions (Ca2+) is one of the most rapid cellular responses to a variety of stimuli, yet obtaining accurate data on the dynamics of intracellular calcium is a major challenge. Historically, specialised liquid handling and fluorescence microplate reading systems have been used to examine bulk population behavior of cells during investigations of the rapid responses of intracellular Ca2+ to various stimuli in vitro. However, the use of a flow cytometer, which operates with an open, as opposed to a pressurized, fluidics system allows continuous monitoring of cells upon the addition of test compounds, providing a method for highly accurate, dynamic calcium measurements (Fig. 2)1. With conventional flow cytometry, the run has to be halted, the sample tube opened and agonist added, then the tube resealed to recommence data acquisition, which adds a gap, or blind spot, in the data.
Cell Cycle Analysis and Ploidy
Plant nuclear DNA content (ploidy) varies over extreme ranges and quantitative measurement of the characteristic 'C-value' is achieved by detecting propidium iodide fluorescence using flow cytometry. Nuclear DNA measurements are often hampered by excessive cellular and sub-cellular debris and autofluorescence from other prevalent cellular components, such as chloroplasts. With over 6 decades of digital signal linearity, the Accuri C6 is the first flow cytometer to cover the full biological range of flowering plant genome sizes from 0.32 to 80.9 picograms in a single run (Fig. 3)2.
Cell Counting
A recent study explored three cell counting applications for viability determination for cultured cell lines, platelet counts in whole, unlysed human peripheral blood samples, and B- and T-cell concentrations in human peripheral blood. The study showed that a flow cytometer with traditional laminar flow fluidics and direct volume measurement capabilities is equally accurate and more precise, than either hemacytometer or counting bead methods for determining cell concentration3. The most precise measurement was obtained by direct volume measurement with the flow cytometer (average CV = 2.1%), with p values of 0.002 and 0.010 when compared to the hemacytometer and counting bead methods, respectively (Fig. 4).
Enter √ at www.scientistlive.com/elab
MaryAnn Labant is with Accuri Cytometers, Ann Arbor, MI, USA. www.AccuriCytometers.com
References:
1. Vines, A. Blanco Fernández and G. McBean (2009). A Flow Cytometric Method for Continuous Measurement of Intracellular Calcium. Irish Flow Cytometry Meeting Poster.
2. Galbraith DW (2009). Simultaneous flow cytometric quantification of plant nuclear DNA contents over the full range of described angiosperm 2C values. Cytometry 75A:692-698.
3. Rogers C., Dinkelmann M., Bair N., Rich C., Howes G., Eckert B. (2009) Comparison of Three Methods for the Assessment of Cell Phenotype, Viability, and Concentration in Cultures and Peripheral Blood. ASCB Poster.
