Ifnγ capture assay kit

In order to determine whether the mRNA we were detecting correlated with the cells producing the protein, PBMCs were stimulated overnight with either HIV-Gag peptide pools, SEB or PMA/Ionomycin. Cells were then labeled with IFNγ catch reagent using the Miltenyi IFNγ capture assay kit and protocol. Cells were allowed to secrete cytokines for 45 minutes before commencing staining. Cells were stained with viability dye and surface antibodies [CD3-BV711 (5 µl per 100 µl of sample, clone: OKT3), CD4-BV421 (5 µl per 100 µl of sample, clone: OKT4), CD8-V500 (5ul per 100 µl of sample, clone: SK1) and CD69-BV650 (5 µl per 100 µl of sample, clone: FN50)], and the flow-FISH was followed. After completing washes outlined at the end of the protocol, cells were labeled with anti-IFNγ PE detection reagent following the Miltenyi protocol. The PE detection reagent was added after the flow-FISH protocol was completed because, if added before the fixation and permeabilization steps, the methanol would have degraded the protein dye and no IFNγ protein signal would have been observed. Cells were then analyzed on the LSR Fortessa.

Humanized mouse splenocytes or human PBMCs were stimulated with PMA (50ng/mL) and Ionomycin (500ng/mL) (Sigma) for 5 hours in the presence of Brefeldin A (Biolegend). Intracellular staining was then performed to detect human NK cell IFN-γ production. In some studies, human NK cells from human peripheral blood and humanized mouse spleen and bone marrow were co-cultured with pig lymphoblasts or K562 cells at a ratio of 1:1 for 4 hours and human NK cell IFN-γ production was determined using an IFN-γ capture assay kit (Miltenyi Biotec).