How To Do Flow Cytometry?
Written by Tim Bushnell, PhD
Flow cytometry is a powerful tool for asking and answering questions at the whole cell level.
The first step in any flow cytometry experiment is to define the hypothesis or biological question that is to be answered. This helps ensure that flow is the correct technique for answering the question. One major determination that needs to made is will the cells be purified (via sorting) or is the experiment just analytical.
The second step in any flow cytometry experiment is to identify the reagents needed for the experiment. In immunophenotyping experiments, this involved knowing what subpopulations that need to be identified. This will lead to determining what antibodies are needed for the experiment. Not all experiments will involve a complex phenotyping – for example, cells may be transformed to express GFP (or other fluorescent proteins) as a measure of the expression of some gene. Other experiments may involve measuring biological processes (reactive oxygen, proliferation, cell cycle etc.) Whatever the experiment, knowing what reagents are needed is the second critical step in flow cytometry experiments.
The third step in a flow cytometry experiment is to understand the output data and how it will be analyzed. This involves understanding the statistical analytical process that will be used to prove or disprove the hypothesis determined in the first step.
The fourth step in a flow cytometry experiment is conducting the experiment. This involves knowing the protocols that will be used. This involves knowing how to setup the instrument and collect the data. This involves knowing how to analyze the data. These are big areas to understand and taking advantage of the expertise such as the Core Manager and staff who run the facility. Expert Cytometry can also help with these issues.
The fifth step in a flow cytometry experiment to analyze the data. This will involve using the flow cytometry software package of choice to identify the populations of importance, as described in step 2. From there, extracting the necessary information to bring to the statistical analysis package will allow for the testing of the hypothesis and discovering the answer that to the question posed in the first step.
The last step in a flow cytometry experiment is to relax and enjoy. Flow cytometry is a powerful tool for answering biological processes at the whole cell level. A well designed and executed experiment can provide a wealth of data, and being the first to unravel some secret about the biology of an organism is a very powerful feeling. Repeat as needed.
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