I did not do FMOs as I did not have enough cells (using brain tissue here). Ive done isotype controls as well however they are have positive staining which could have possibly been due to an improper match of the isotype control with the antibody. I know unstained corrects for autofluorescence but I was wondering if I could use it to also gate for my negative cell population?
The fact that your isotype controls are positively staining suggests that you may have non-specific binding of your antibody. What are you using to block before antibody staining? As per your question, the isotype controls are traditionally required to define your negative cell population and would be particularly important in doing so given that they appear to be "positive staining". The unstained controls are more important for color compensation set-up.
Hi Lavi Doray
Of course you could. whether the isotype control is better we don´t know. Some researchers don´t use isotype control, but, if you want to publish your results I think you will need it.
Using the unstained sample to set a region for the cells that are not stained in your sample (=negative) can be fine but this depends on how your controls / isotype staining looks like e.g. how much shift in fluorescence intensity you get/ is this even over the whole population (assuming your sample prep has not only one type of cells) and what you will actually want to do with the resulting data.
Using unstained cells is often the best option but can critisized by referees. I propose to try several blocking reagents such as BSA or hyper-immunoglobulin. Often there is a high autofluorescence still in the negative population so that you have an overlap with the positively stained cells after using isotype controls
You probably ran single stains at one time to set up the compensations requiered for your full staining. This can also give you a general idea if negative controls (with the defined compensation from your single stains) can be used as negative control of your fully stained samples. Unless a single color still results in a shift in another color when the compensations are applied, I would think the non-stained negative controls can be used for the definition of the negative population in a given color. Moreover if the fluorescence shift is important between the negative and the positive populations. If you only observe a slight shift, then make sure that this slight shift is absent from all of your single stains when analyzed with the same compensation matrix as your fully stained samples.
Hope this helps.
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