Flow Cytometry: Tips for Fixation & Permeabilization | CST Tech Tips
Advice on sample preparation to improve your flow cytometry analysis of intracellular targets, focusing on the fixation and permeabilization steps.
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Transcript:
What do I need to know about fixation and permeabilization and the protocol for flow cytometry? Hi, I'm Rob, Senior Research Associate in the Flow Cytometry Group at Cell Signaling Technology, and this is CST tech tips.
Flow Cytometry can be performed with live cells if all of your targets are expressed on the extracellular surface. But when you're targeting intracellular proteins, the cells will need to be fixed, permeabilized and immunostained with antibodies and/or conjugated antibodies in that order.
Fixation cross-links macro-molecules in the cells and locks the target proteins in place, and permeabilization allows antibodies to access the intracellular space. Here are some considerations to keep in mind when performing a fix-perm protocol. First, you can find the recommended fixation and permeabilization protocol for each flow validated antibody on https://cellsignal.com. Just type the product number in on the search bar to bring up the product page.
Prior to fixation, your cells need to be in a single-cell suspension to ensure accurate readout and analysis. If you're starting material is adherent cells or tissues, you'll need to thoroughly dissociate the cells with trypsin or other proteases. Once you have your single cell suspension, you can proceed with fix, perm, and staining steps in individual flow tubes, or you can fix and perm large batches of cells and aliquot them for subsequent analysis.
For many antibodies, the recommended fixative is formaldehyde, the aqueous monomeric form of formaldehyde. Fixing the cells prior to permeabilization helps to minimize loss of target proteins and preserve signaling states. This is particularly important when characterizing signaling activities, for example, with phospho-specific antibodies.
After fixation, ensure the fixative is thoroughly washed out. This avoids excessive crosslinking of proteins within the cell and eliminates the risk of antibodies being crosslinked to non-specific targets by leftover fixative. Once the cells are fixed and washed, they're ready to be permeabilized.
Some antibodies require use of a specific permeabilization reagent, while others work equally well with multiple reagents. Again, you can find the recommended permeabilization reagent in the antibody product page on https://cellsignal.com.
The majority of our flow validated antibodies against intracellular targets can be used with methanol permeabilization. Solvents like methanol can provide more access to the interior of organelles such as nuclei, mitochondria and lysosomes. In certain cases, methanol affects the structure of the epitope. This could positively or negatively affect detection depending on the antibody and epitope. Detergents likes saponin can better preserve the native structure of epitopes but may not provide as much access.
After your cells are fixed and permeabilized, you're ready to proceed with antibody staining steps.
Thanks for watching. If your flow experiment will incorporate both surface markers and intracellular targets, check back for upcoming video on multiplexing. If you found this video helpful, please hit the like and subscribe buttons for more Tech Tips. Anytime you have a question about an antibody or protocol, get in touch with the scientist at https://cellsignal.com/support. Good luck with your experiments, and we'll see you next time.
👉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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