Anti cd107a apc antibody

MaxiSorb 96 well flat bottom plates (Nunc, Thermo Fisher Scientific, Waltham, MA, USA) were coated with 10 µg/ml PBS of the indicated antibodies for 16 h at 4°C and subsequently blocked by incubation with 10% FCS for 20 min. Splenocytes isolated from mice were added to the plates and incubated with 2 µg/ml anti-CD107a APC antibody and GolgiStop (BD Biosciences) for 4 h at 37°C. Stimulation with 25 ng/ml PMA plus 500 ng/ml ionomycin (both Sigma-Aldrich) served as positive control. Subsequently, splenocytes were stained with both anti-CD3-PerCP and anti-CD49b-PE, permeabilized, and stained with anti-IFNγ-FITC as described for the intracellular staining.

Flow cytometry was performed using a NovoCyte flow cytometer (ACEA Biosciences, Inc., San Diego, CA, USA). For PD1 expression analyses, mGFP or PD1-mGFP transduced MSCs were labeled with anti-PD1-APC antibodies (Miltenyi Biotec, Bergisch Gladbach, Germany). For CD107a expression analysis, HuCCT1 cells were pretreated with 1 × 10¹⁰ control CDNVs and PD1+ CDNVs for 3 h, followed by co-culture with T cells or NK cells for 6 h at a 5:1 or 10:1 effector to target (E:T, immune cell: HuCCT1 cell) ratio in the presence of GolgiStop (BD Bioscience, Franklin Lakes, NJ, USA) and anti-CD-107a-APC antibody (BD Bioscience, Franklin Lakes, NJ, USA) in RPMI 1640 medium. The immune cells were collected and stained with anti-CD8-FITC or anti-CD56-FITC (Miltenyi Biotec, Bergisch Gladbach, Germany) for NK and T cells, respectively, prior to flow cytometry. Cytometry data were analyzed using FlowJo v10.8.1.