[Cytometry] Antibody titration & staining index

Barsky, Lora lbarsky at med.usc.edu
Mon Feb 17 10:40:16 EST 2014

I've always thought that homogenous populations like monocytes could be properly titrated by preparing two groups of tubes, one group stained with a serial dilution of CD14 (8 tubes) and another group stained with a serial dilution of isotope control (8 tubes).   It is critical that the two antibodies, CD14 and isotope control, be matched isotype class and have an identical beginning concentration.  The tubes are stained and washed separately. 

After processing, the matched pairs are combined (16 tubes reduce to 8 tubes) and acquired.  Also acquire an unstained sample.  The unstained sample should be processed as the others were, except no antibody is added.  This will introduce the same artifact to the negative which may now be present in the antibody labeled tubes.  Artifact may come from processing reagents. 

Now your data set contains positive and negative populations and a stain index or  alternatively a signal to noise ratio be calculated. The negatives now show at what concentration the cells bind to excess antibody concentration while the positives will show decreasing intensity of label.   The point at which the negatives and positives show the largest separation is chosen as correct titer.

Remember not to use lymphocytes as your internal negative.  They and monocytes have differing autofluorescence intensities, therefore become inadequate pairs for determining stain index or signal to noise ratio.   Also, monocytes have been shown to bind with higher background fluors containing Cy- fluors, but I figure you know this already.  Choosing to correct this issue by finding staining tigers. 

Best wishes, 
Lora Barsky
Director, USC flow cytometry core facility
University of Southern California

Sent from my iPhone

Sent from my iPhone

> On Feb 17, 2014, at 6:41 AM, "Ina Laura Pieper" <inalaurapieper at gmail.com> wrote:
> 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,
> http://ucflow.blogspot.co.uk/2009/06/antibody-titrations.html
> 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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