4 situations where Flow Cytometry can help | CST Tech Tips
Should you switch from western blot to flow? We’ll go over situations where flow cytometry can take your research further.
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Transcript:
When and why should I consider flow cytometry, and what kind of information can flow cytometry experiments give me, that western blots can't? I'm Sarah, Development Scientist at Cell Signaling Technology, and this is CST Tech Tips.
Western blotting is used to detect proteins and analyze their expression using antibodies. But western blotting does have its limitations. Flow cytometry also employees antibodies, but it uses florescence to detect and analyze protein expression on a cell-by-cell basis. Here are four situations, where it can be worth going with flow over western blot.
Situation one, you want to analyze multiple targets in the same sample in a shorter time. Multiple targets means using multiple antibodies. To do this with westerns, it's possible to strip and reprobe the blot, or set up multiple blots. But reprobing adds a lot more time to your work flow, and running blots in parallel requires more lysate. Multiplexing antibodies with flow saves time, and you can get more data from your samples.
Situation two. You want to analyze specific cell populations, or subpopulations. This could apply if your input material contains cell types, for example tissue or immunological samples. Flow cytometry allows pheno-typing of populations and sub-populations by employing antibodies to proteins selectively expressed in each cell type. Because of this, flow cytometry is widely used in studies of immunology, cancer biology, developmental biology and more.
Situation three. You want more sensitive detection of intracellular signaling events. Let's say you are measuring a signaling response to a stimulus, but only a portion of the cells in your sample are responding. This is not uncommon for many signaling readouts.
In western blot, lysates are pooled into one sample, so your signal from the responding cells will be effectively, diluted out. But in flow cytometry, analysis is conducted on a single-cell basis, enabling you to detect and quantify these rare signaling events, and define the cell types in which they occur.
Situation four. You want to analyze multiple readouts and look for correlation between them. For example, if your hypothesis is that two subpopulations will respond differently to a treatment, you can design an antibody panel including antibodies to multiple phenotypic surface markers to gate your populations, and antibodies to evaluate expression or activation of signaling proteins. While multiplexing may require optimization, the benefits of this multi-parametric analysis can be a very powerful tool to gain biological insights.
Once you've decided to add flow to your experimental tool box, here are two steps to take next. First, you'll have to arrange access to a flow cytometer, either through your institution's core facility or a collaborator. Second is to start designing your antibody panel, keeping in mind the antibody you used in your westerns may not perform in flow. So when you're selecting antibodies and antibody conjugates, make sure to look for validation data performed specifically in flow cytometry.
If you found this video helpful, hit the like and subscribe buttons, and share with your colleagues. You can find application specific protocols under the antibody product pages on http://cellsignal.com. Any time you have a question about an antibody or a protocol, you can ask one of our scientists at http://cellsignal.com/support. Good luck with your experiments!
👉About CST: Cell Signaling Technology (CST) is a private, family-owned company, founded by scientists and dedicated to providing high-quality research tools to the biomedical research community. Our employees operate worldwide from our U.S. headquarters in Massachusetts, and our offices in the Netherlands, China, and Japan. http://cellsignal.com/about
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