Biomedical Engineering Reference
In-Depth Information
against which a screen is designed, and then screen their compound deck for hits
against this target. The second step is usually the desire to characterize the resulting
hits to identify practical lead candidates that may be chemically tractable or otherwise
amenable to analogue synthesis. And the third step is an understanding of the
effects these chemical hits may have on cells before embarking on an extensive and
expensive analogue campaign. It is especially interesting to characterize up front
some of the specific interactions that these compounds may have on cells where the
final target is present.
Use of analytical cell cycle profiles in the evaluation of compounds were
considered until recently only a secondary step in the selection of compounds and
not an appealing screening tool due to the low-throughput attainable by flow
cytometers. The rate-limiting step was sample acquisition. Lately, the capacities of
the instrumentation have been expanded to include plate collection systems that speed
the process considerably. Included with these advancements are some additional
systems, which allow today up to 386-well plates to be handled for sample acquisi-
tion [6]. This advance demonstrates that change is happening to flow cytometry and
more “screening-like” approaches are just around the corner. The limitations now
seem to be more related to what information a sample can provide, such as what
number of cells and how many parameters per cell does one need to get a reasonably
interpretable answer to an experimental question.
5.2.2 The Cell Factor
Obviously, the most important factor to be considered when using flow cytometers to
conduct a study of the interactions of compounds or macromolecules with cells is the
health of the cells. One key element for healthy cells, and hence reliable results, is that
cell culture conditions need to be stringently controlled to limit the variability in
the response of cells to drug stimulation. For example, overgrown cultures or cells
just out of a stress condition (frozen storage) provide unreliable sources for
compound evaluation.
Also, when looking at the effects that compounds may have on cells, the
investigator should also pay particular attention to stress conditions that may be
induced by a compound or even the carrier used to solubilize the compound during
the evaluation process. This concern becomes especially relevant when addressing the
advancement of anticancer agents because of their potential to alter specific events
occurring during cell cycle traverse. Likewise, some organic solvents, such as
dimethyl sulfoxide (DMSO), formamide, and others are capable of inducing cell
differentiation at subtoxic doses [7].
5.2.2.1 Sample Processing for Cell Cycle Analysis Samples for cell cycle
analysis from cell culture or animal tissues are based on their DNA content and
can be processed to either obtain as individual nuclei (this is usually important when
studying ploidies) or as whole individual cells that can allow other measurements,
such as associated cytoplasmic marker quantifications. Also, when collecting whole
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