18 ANALYTICAL AND LABORATORY EQUIPMENT
Multi-parametric cellular analysis
Flow cytometry offers several advantages over other techniques by enabling multi- parametric, individual cell analysis. MaryAnn Labant reports.
A
ccumulating, 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. Te ideal circumstance would be to have an instrument, which could perform a variety of multi- parametric applications.
Fig. 2. Dot plots of continuous calcium dynamics, obtained on the Accuri C6, clearly indicate the absence of gaps on addition of test compounds. Fluorescence of Fluo-4 and Forward Scatter versus time1
.
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,
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Fig. 1. Just 18 inches wide and 17 inches deep, (36.3 x 41.9 cm), the 30lb (13.6 kg) Accuri C6 Flow Cytometer fits on any lab bench and features 2 lasers and 6 detectors.
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
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