Stimulation diminishes non-specific binding of tandem dyes

In Fig. 2, human PBMC were split into two conditions, unstimulated (Fig. 2A-F) or a 6 hr stimulation with the Cell Activation Cocktail of PMA/Ionomycin with Brefeldin (Fig.2G-I). After stimulation, both conditions were stained with the antibodies listed in Table 2 with appropriate fixation and permeabilization for the detection of intracellular cytokines. Staining cell surface markers in the presence of the True-Stain Monocyte Blocker™ effectively blocked any non-specific binding due to cyanine-based fluorophore tandems. 

The acceptor molecule of PE/Dazzle 594™ is not cyanine-based and exhibited no non-specific binding under either of these sample preparation conditions, whether the cells were stimulated or not. This fluorophore can exhibit some non-specific binding when used only in a 2-color panel, but does not exhibit non-specific binding when used to stain PBMC, LWB or pre-lysed blood in a multicolor panel with other tandem dyes. On the other hand, APC/Fire™ 750, APC/Cy7 and PE/Cy7, all of which are derived from the cyanine family of fluorophores, have variable non-specific binding in multicolor panels on different donors and with different sample prep conditions.

Description Clone
CD8 BV510™ SK1
CD4 BV650™ RPA-T4
CD19 PE/Dazzle™ 594 HIB19
CD3 APC/Fire™ 750 UCHT1
CD14 BV421™ HCD14
CD56 PE/Cy7 5.1H11
IL-4 Alexa Fluor® 647 MP4-25D2