When I used an antibody, I always do the titration two folds up and down from the manufacturer's recommendation. I have used MFI (median fluorescent intensity ) and the histograms to analyze my titration data. In a titration, I only use one antibody at a time and since it does not require FMOs, I gate my population purely based on the unstained population. If I take titration results as one data point, it makes massive changes and bias because of the different controls and the different gating strategies. As an example, if one antibody is stained 100% with the antibody during the titration, during the actual protocol, I get less than 1% staining with the same antibody. What I think is during the actual protocol, since I am using multipanel fluorochromes, the other antibodies may be interfering with the expression of the specific antibody.(This is why I am using FMOs to minimize the florescent spillage). Do you think it is wise to take my titration data as one data point with my actual flow protocol data points?
Hello,
I think it is great that you are doing titration experiments before you dive into the real deal. Usually, the antibody concentration in commercially available products is more than enough for a real good binding.
However, titration and FMOs mai share some common aspects, but they are used for different things.
- Titration is usually performes in order to find the concentration at which your cells are best stained or find the balance between satisfactory staining and antibody saving. From my experience, i can tell that for some very well represented surface antigens like CD3 on T-cells, you can go way below the manufacturer's recommendation and still have a very good staining (as seen during flow cytometry; the provider was BD Biosciences which has excellent antibodies)
-FMO are not used to minimize the spillage. Unfortunately, you can't do that in a cytometer. If you want to minimize the spillage, then you must carefully select the fluorophores your antibodies are labeled with so their emission spectra do not overlap. However, if you have a panel with many distinct fluorophores, spillage will occur as you are forced to overlap some emission spectra. That is where FMOs come in handy. They do not reduce the spillage, but they serve as a mathematical model for the cytometer's software in order to compensate the spillage, that is the software will do the maths and show some data that is partially mathematically rendered.
These being said, i do not encourage you to use the data from your titration experiment as part of your gating strategy in an experimental protocol. FMOs should be enough.
Also, not sure if the example you gave (100% vs 1%) was real, but if it was, then something may not be right. Antobodies have high specificity. Some interferences can occur, but from 100% to 1%, i have never seen something like this with flow cytometry.
Hope this helps!
Thank you so much for your explanation... I will not be taking titration as one point of my data and when I was gating my populations, I have done some obvious mistakes. Will correct my mistakes and your explanation about FMOs really helped! Thank you so much!
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