Foxp3

Titration of FoxP3 (tagged with PE). Note the gradual and slow population shift over increasing doses of antibody.  (UU - unstimulated unstained, US - unstimulated stained, SS - stimulated & stained)

As the dose of antibody is inreased, the entire helper T cell population is taking up the stain (which is unwanted behavior)

There is no drop in median PE expression with doses upto 10 microL. This means there might be yet more antibody targets left to bind

In this case, 2.5 miroL dose which gives good positive population (2.86%) without staining the negative population. So we select this dose.

Antibody clone: 236A/E7, FoxP3 - PE

Transforming growth factor beta 1

Titration of anti TGFB1 antibody

Without stimulation

Titration plots for increasing doses of anti-TGFB1. Note the vertical line drawn at unstained (UU) and successive population shifts (non specific binding?). The ideal dose in this case must be close to 0.625 microL.

Doses ranging from 0.625 to 10 microL; note that entire cell population is moving

Plotting median FITC versus dose shows a continuously increasing trend. so no help from there!

With antigenic (MTb) stimulation

Does not make much of a difference

Thus, still undecided on the dose.

(Antibody clone TW4-9E7 bound to Alexa fluor 488, detected on FITC channel; cells - healthy peripheral blood mononuclear cells).