[Cytometry] Antibody titration & staining index

Ina Laura Pieper inalaurapieper at gmail.com
Sun Feb 16 15:39:12 EST 2014

Hello flowers,

What's the objective way of deciding on the optimal antibody concentration
during antibody titrations? I've searched and found this description on the
UC Flow blog of plotting the stain index against antibody concentration,
However, to calculate stain index it seems I need a positive and negative
population in my sample, probably of the same autofluorescence, which isn't
always achievable.

What do I do if I want to calculate the optimal concentration in a
titration of a CD14-antibody in whole blood? I've done overlay histograms
of stained and unstained samples gated on monocytes (positive) and
granulocytes (negative), so that i can visually identify the concentration
at which the granulocytes are no longer staining unspecifically. Is this
the right way to do it? Seems very subjective to me. From the attached
figure I think a 1:5 dilution is the best (no unspecific binding on
granulocytes whilst still a clear monocyte signal).

What should I do when staining a homogenous sample, and there is no
negative population?

Best regards,
Ina Laura

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