Apc conjugated anti human cd69

The following primary antibodies were used in immunoblotting and flow cytometry: rabbit anti-GAPDH (Santacruz), rabbit anti-phospho-p70 S6 kinase (S6K) (Thr 389) (Cell Signaling), rabbit anti-p70 S6K (Cell Signaling), and APC-conjugated anti-human CD69 (Biolegend). Anti-human CD150 antibody clone 5C6 was generated in our laboratory (Erlenhoefer et al., 2001 (link)). The following labeled secondary antibodies were used: Alexa488-conjugated anti-mouse (Life Technology), Alexa594-conjugated anti-rabbit (Life Technology), and horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Cell Signaling). For flow cytometry 2 × 10⁵ cells per sample were stained with respective antibodies in FACS buffer (PBS containing 0.4% bovine serum albumin (BSA) and 0.02% sodium azide). Dead cells were stained with PI (Biolegend). Cells were acquired immediately using a LSR II flow cytometer (BD) and the data were analyzed using FlowJo (Cytek Development) software.

To selectively monitor the activation of T cells, the purified T cells were labeled with carboxyfluorescein succinimidyl ester (CFSE, Biolegend) as per manufacturer's protocol, and further incubated with target cells in the presence of bsAbs or parental antibodies. After 24 h, cells were labeled with pERcp/Cy5.5-conjugated anti-human CD25 and APC-conjugated anti-human CD69 (Biolegend), and analyzed by flow cytometry. The release of IL-2, IFN-γ, and TNF-α in the cultured supernatant was measured by enzyme-linked immunosorbent assay (ELISA) kit (Thermo Fisher Scientific). Results are shown as mean of duplicated samples ± SD.