Clone selection...
Ensure antibody clone / manufacturer differences are tested when selecting antibodies for a panel.
a)
b)
(a) Two clones were available for the transcription factor PU.1 (found in some myeloid cell subsets). Both are on the same fluorophore, but only clone 7C6B05 was able to resolve a separate population. A titration was preformed, but the population was never discernible by clone 7C6B05.
(b) Here the same clone from two different companies on two similar fluorophores was evaluated on the same PBMC donor. You can see company A conjugated-antibody was able to resolve a CD3+ CRTH2+ population, however company B’s conjugated antibody was not. Increasing the concentration did not help and simply resulted in more spread in the negative.
Viability titration
Benefits of titrating not only antibodies but viability dyes.
(above) Titration revealing populations that are unresolvable with an abundance of antibody due to non-specific binding.
(below) Often overlooked, titrating the amine-reactive viability markers can result in tighter negative populations due to minimised unbound fluorophores and baseline restoration, which has less of an effect on other important markers even in the negative (live) population.
Annotate your experiment before or during data acquisition
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