Can I compensate with beads for my experiments. I use 4 colours in a single tube (FITC, PE, PerCP Cy5.5, APC). What does it matter if I use beads for my compensation. Why can't I just use beads to compensate? Or how would compensating with cells improve my data? (P.S. anyway compensation deals with cutting off percentages of emissions from possible overlapping fluorochromes, so it is just the property of light, how would it matter if cells autofluoresce or not?)
Hi Sam,
Because fluorochrome spillover between channels is completely independent of the autofluorescence (AF) of your compensating particle, compensation using beads (eg BD comp beads) is much better than using cells under almost all circumstances.
The only relevant consideration with AF with respect to compensation is whether the specific "positive" and "negative" single stained control particles/cells have identical AF. This is hard to determine in a mixed biological population.
Beads have highly defined/uniform autofluorescence (AF) whereas individual cell populations can have distinct patterns of AF over a range of emission channels. This means that if using cells for single stained compensation controls, you would ideally gate your stained population initially based on scatter/AF and only then would you look for positive/negative flourochrome staining (so that the neg and pos cells have the same AF). This is not necessarily trivial, or even possible for all populations/Ab combinations. If you are interested in lymphocytes, for example, you can probably do this quite easily, but most myeloid populations (eg monocytes, neutrophils eosinophils and tissue resident macrophages) are a different matter have distinct patterns of AF.
Bottom line...use beads wherever possible!
Hi Sam,
yes you can use beads only, but in my experience, if you use cells like monocytes or dendritc cells, you'll get better compensation values if you stain these cells with Abs conjugated with the different "colours" you'll use. Autofluoresce can be seen not just in the Forward and Side scatter PMTs but also in the others (FL1, FL2...) therefore you need to compensate this "contribution" to the signal.
Hi Sam,
Because fluorochrome spillover between channels is completely independent of the autofluorescence (AF) of your compensating particle, compensation using beads (eg BD comp beads) is much better than using cells under almost all circumstances.
The only relevant consideration with AF with respect to compensation is whether the specific "positive" and "negative" single stained control particles/cells have identical AF. This is hard to determine in a mixed biological population.
Beads have highly defined/uniform autofluorescence (AF) whereas individual cell populations can have distinct patterns of AF over a range of emission channels. This means that if using cells for single stained compensation controls, you would ideally gate your stained population initially based on scatter/AF and only then would you look for positive/negative flourochrome staining (so that the neg and pos cells have the same AF). This is not necessarily trivial, or even possible for all populations/Ab combinations. If you are interested in lymphocytes, for example, you can probably do this quite easily, but most myeloid populations (eg monocytes, neutrophils eosinophils and tissue resident macrophages) are a different matter have distinct patterns of AF.
Bottom line...use beads wherever possible!
I agree with Sam. The only time I use cells for compensation is when the dye is not conjugated to an antibody (i.e. Rhodamine 123, or Sytox Blue).
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