There are a few factors that get evaluated when designing immunophenotyping panels.
- Antigen density
- Brightness of fluorophore
- Fluorophore overlap (Compensation)
- Expression of protein (eg. is antigen A also expressed on the same cells as antigen B)
It takes a lot of experience to be able to put these all together. Over the last little while Tessa has been comparing a bunch of antibodies in order to help us have a more quantitative answer for “brightness of fluorophore”. She obtained roughly 45 of the same CD4 antibodies, but conjugated to different fluorophores. CD4 is relatively conserved from person to person (~50,000 molecules per cell) and can be useful for a longitudinal study like this. Below is a chart of Stain Index for each of these colors. Stain index is just a fancy way of calculating how separated the positive cells are from the negative cells. Most of it makes sense if you’ve paid attention to this kind of thing. One stand out is that BB515 is much brighter than FITC. Probably more expensive too but… We can and will use this data to also give us a more comprehensive understanding of “fluorophore overlap” but that still needs a bit of polishing. If you would like to play around with these FCS data files just let us know and we will send you a link to ubox.
Click on chart to enlarge