To compare positive and negative cell populations in flow cytometry, should I compare unstained cells with antibody stained cells? Or with the isotype control?
Most papers show comparison with isotype control, but I am getting high background from my isotype control(another issue for another question I guess).
But first I want to know what is the common practice. I am afraid to base my results of positive cells with unstained controls and later on it might be rejected by journals.
.
Isotype controls are most informative. The reason you would not use unstained ctrl (I assume that by this you mean FMO ctrl rather than completely unstained) is that that way you can not rule out non specific binding of the Ab coupled to a fluorophore in question. If you have a very high staining with your Isotype control, consider optimising your Fc receptor blocking (if you are trying to stain a surface marker, not the intracellular markers of course) or alternatively your compensation and spillover of other colours you are using in your panel, into the channel that detects your fluorophore of interest.
Though not always possible as a control (because manufacturers don't always specify which antigen was used) for directly conjugated antibodies a blocking peptide or recombinant protein should reduce your positive signal if real.
Hello Adamska Mr
You will have to use the isotype control.
The fluorochrome-conjugated primary antibody will bind to the antigen specifically and will result in positive staining. However, the Fc region of your primary antibody will bind non-specifically to the Fc receptors resulting in non-specific binding and therefore background fluorescence. Additionally, it may produce other non-specific protein interactions, which will also result in background florescence. So, you will have to estimate the level of background staining.
For this purpose, you will have to use a fluorochrome conjugated non-specific antibody, which consists of Fc region of the host species and with the same isotype as the primary, which is termed as the isotype control antibody. The level of fluorescence resulting from staining with this isotype control will be the background fluorescence.
Please note that the isotype control should match with the primary antibody with respect to the host species and the isotype. Also, the conjugated fluorophore and the concentration of the isotype control used for staining should match with the primary antibody.
As far as high background from your isotype control is concerned, there are many factors involved like quality of the isotype control antibody namely, the quality of the conjugation. Poor conjugation can lead to high background as the loosely bound dye molecules can associate with other components in the sample. So, ensure that the antibody is of high quality. Optimize the concentration of the antibody as high concentration of the isotype control antibody can lead to high background. Optimize the washing and blocking steps as inadequate washing or blocking can leave unbound sites available for non-specific binding of the fluorescently tagged isotype control.
Best.
David G Spiller Thank you for your explanation. Much appreciated.
Malcolm Nobre Thank you so much. You guys are what make this platform thrive. I am very grateful.
For start, it very much depends on what cells are you staining and what's the protein target you're staining for. In theory, we should always use isotype control stained samples for comparison and voltage setup, but in practice most of the times we don't, we just use unstained samples for control, because it frankly isn't necessary. Maintaining a panel of antibodies of various species and isotypes, labeled with various fluorochromes just for isotype control stainings can be costly and most of the times just makes no economical sense. The use of Fc blocking reagents (antibodies that block Fc receptors) before staining with your antibodies is almost always enough to eliminate non-specific signal. If you're seeing high background from your isotype control, then you're most likely using too high antibody concentration. If you add too much of whatever antibody to cells, you'll eventually get a signal. In flow cytometry, while most people don't bother titrating their antibodies, they can be used in 10-100x lower concentrations than the manufacturers suggest and still give very good signal. That said, you need to know something about your protein of interest, its expression levels, etc. - look for some literature data. If your protein of interest is intracellular - yes, you'll most likely get a lot of background and will need several controls, but for surface receptors good antibodies are usually specific enough. Also, if you don't have in your cell population a lot of B cells or granulocytes, you don't need to worry about isotype controls. In the end, if you feel confident your stainings are specific, most journals wouldn't ask for isotype controls for everything.
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