Why use isotype controls

One alternative to an isotype control is the isoclonic control. This is where cells are stained in the presence of an excess of identical unlabeled antibody. The unlabeled antibody takes up all the binding sites, preventing the labeled antibody from binding specifically. Thus any signal that is detected must come from non-specific binding. You can create and edit multiple shopping carts Edit mode — allows you to edit or modify an existing requisition prior to submitting.

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Third-Party Cookies being blocked. Please Note. Search Thermo Fisher Scientific. Search All. Isotype Control Antibodies. See Navigation. Have you tried adding excess immunoglobulins or whole serum to your buffer? Each of these can help pull down your nonspecific adherence. Are you certain it is non-specific antibody adherence you are are dealing with and not free fluorochrome adherence? You can find out by using an isoclonic control. If you add massive amounts of non-fluorochrome conjugated monoclonal antibody to your staining reaction, your fluorescence should drop.

If it does not, your issue is not due to nonspecific antibody binding, but to free fluorochrome binding. For cell signalling and cytokine staining, besides the FMO control, make sure not to neglect a biological negative control, whether that be unstimulated cells, or cells treated with an inhibitor of phosphorylation. With the proliferation of these dyes in different colors and for both viable and fixed cells, there is no reason to not use these dyes.

Viability dyes are essential for removing dead cells that will non-specifically uptake antibodies. In conclusion, isotype controls are useful for demonstrating that there was poor blocking of the cells. They should never be used to determine or set positivity in fully stained samples. To learn more about which controls you should use for your flow cytometry experiments and to get access to all of our advanced materials including 20 training videos, presentations, workbooks, and private group membership, get on the Flow Cytometry Mastery Class wait list.

Sun Tzu was a Chinese general and philosopher. So I have identified 5 points that I think lend themselves to thinking about flow cytometry. The longer the cells are out of their natural environment, the less happy they…. Mastering foundational concepts are imperative for successfully using any technique or system. Ever since then it has made its way into popular culture.

To Grok something is to understand it intuitively, fully. As a cytometrist, there are several key concepts that you must grok to be successful in your career. These foundational concepts are the key tools that we use day in and day out to identify and characterize our cells of interest. Cells Flow cytometry measures biological processes at the whole cell level. To do…. Fluorophore selection is important. I have often been asked by my facility users which fluorophore is best suited for their experiments.

Once you have narrowed down which fluorophores you can excite and collect the correct emission, you can further refine the specific fluorophore that is best for your experiment. In this blog we will discuss how to determine what can work with your microscope, and how…. It is not possible to predict the unspecific binding of a primary antibody to non-targets molecules, as such, an isotype control is necessary in order to evaluate the contribution of non-specific background signal to the staining results and to distinguish between non-specific and specific antibody staining.

Generally, you can have a background signal when your primary antibody binds non-specifically to the Fc receptors expressed on cells, such as: B cells, macrophages, monocytes, and dendritic cells. Additionally, the primary antibody can also bind non-specifically to lipids and carbohydrates and can interact with off-target proteins or debris present in the sample. An isotype control antibody must match the properties of the primary antibody being used.

It must be raised in the same host species, be of the same isotype and Ig subclass, and have the same conjugation. It is also important to use an isotype control from the same supplier as the primary antibody, as the isotype control should ideally have an equivalent label to antibody ratio with respect to the primary antibody, and conjugation methods and the resulting number of fluorophore molecules conjugated to a particular antibody can vary between suppliers.

You can reduce non-specific antibody binding by blocking Fc receptors, for example, by adding an excess of BSA to the buffer. Why are isotype controls important? What causes non-specific background staining? How to choose an isotype control? What experimental conditions should you consider? How can you reduce non-specific antibody binding?