Flow cytometry can be leveraged to measure intracellular signaling states using antibodies specific for post-translational modifications, including phosphorylation. You can design experiments that incorporate inhibitors, agonists, or antagonists to modulate signaling activities in your samples and test hypotheses about the signaling mechanisms that may play a role in your model system. In this video, Rob and Sarah present five tips for successful intracellular flow experiments.
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Contents:
0:50 - Phospho-Flow: Practical and Informative
1:45 - Importance of Timing
2:12 - Finding the Right Readout
2:49 - Using Downstream Readouts
3:09 - Positive and Negative Controls
Transcript:
How can I use flow cytometry to analyze activation of intracellular signaling pathways? I’m Robert, senior research associate in the Flow Cytometry group.
And I’m Sarah, development scientist at Cell Signaling Technology, and this is CST Tech Tips.
Flow cytometry is ideal for assessing signaling events in biological systems. It provides quantitative multiparametric readouts at the single cell level, with high sensitivity and speed.
You can use flow cytometry to test hypotheses that certain stimuli, such as treatments with inhibitors, agonists, or antagonists, can modulate specific signaling activities. Here are 5 tips to remember when you’re designing your experiments.
#1 Phospho-Flow can be both practical and informative. Phosphorylation events are among the most commonly studied signaling mechanisms, and can be characterized with the use of phosphorylation specific antibodies. Using phospho specific antibodies in flow cytometry is not that different from using antibodies directed against other intracellular targets. Protocols using formaldehyde to fix, and either a detergent or a solvent to permeabilize, are generally compatible with analyzing phosphorylation. Also, phosphatase inhibitors are usually not required, as protein crosslinking by the fixative also stops phosphatase activity. As with total protein targets, the ideal protocol can vary by target and by antibody, so look for the recommended protocol for your CST antibody on www.cellsignal.com.
#2 Timing is key. Protein phosphorylation and dephosphorylation can be transient, and the timing between your experimental stimulus and fixation can affect the magnitude of your readout. If the dynamics of your target of interest are unknown, setting up a time course experiment can be informative. Some targets may be maximally phosphorylated after a matter of minutes, while others may take hours or even days to reach these peaks.
#3 Sometimes, phosphorylation may not be the optimal readout for a signalling assay, as other modifications or even total protein expression levels may be a better indicator of signaling changes. Other post translational modifications including acetylation, methylation, and cleavage can also be analyzed by flow cytometry. For example, methylation specific antibodies are often employed in epigenetics studies, and cleavage specific antibodies can be used to assess apoptotic signaling. Once again, timing can be crucial.
#4 If an antibody to your protein of interest isn’t available to provide a direct readout, you can consider choosing a downstream node in that signaling cascade to use as an indirect readout of that pathway. Remember to look for antibodies and antibody conjugates that have been validated specifically for flow cytometry.
#5 Finally, including a known positive or negative control in your experiment can tell you if there is an issue with your experimental design, or if your antibody isn’t performing as expected. If a control treatment is known to modify the phosphorylation of your target, this can give you an idea of the magnitude of change to expect in the readout of your experimental samples.
Thanks for watching! If you found this video helpful, please hit the like and subscribe buttons for more Tech Tips. You can find full application-specific protocols for CST antibodies on cellsignal.com. Anytime you have a question about an antibody or a protocol, get in touch with a scientist at www.cellsignal.com/support. Good luck with your experiments, and we’ll see you next time.
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