Coating buffer
1X PBS (10X PBS [Roche] diluted to 1X with H2O)
3 μg/mL of IFN-γ capture monoclonal antibody (mAb) and/or 3 μg/mL of IL-2 capture mAb (both from Becton Dickinson/Pharmingen)
The dual coating buffer contains both mAbs, whereas the IL-2 coating buffer contains only the IL-2 capture mAb and the IFN-γ coating buffer contains only the IFN-γ capture mAb. Wells coated with the latter two buffers are used to compensate for the color sensitivity of the CTL analyzer during spot counting. Typically, for wells coated with IL-2 or IFN-γ coating buffers, cells plated in the wells are stimulated with a positive control stimulus (CEF or anti-CD3) in order to obtain a signal suitable for calibration.
Coating buffer should be prepared within a few hours of use.
