Anti cd8 apc

The following mAbs were used for flow cytometry: anti-CD3 PerCP, anti-CD4 FITC, anti-CD4 PerCP, anti-CD4 PE-Cy7, anti-CD8 PErCP, anti-CD8 APC-Cy7, anti-CD28 FITC, anti-CD95 APC, anti-CD195 (anti-CCR5) PE, anti-Ki67 PE, anti-HLA-DR APC, and anti-HLA-DR FITC (BD Biosciences). Additionally, annexin V PE was purchased from BD Biosciences, anti-CD8 APC and anti-CD8 PE were obtained from Beckman Coulter (CA, USA), and anti-CD38 FITC was purchased from StemCell Technologies (Vancouver, Canada). Flow cytometry data were acquired on a FACSAria II (BD Biosciences). All data were analyzed using FlowJo software (Tree Star, Ashland, OR, USA).

Non-adherent allogenic PBMCs were analyzed for intracellular secretion of interferon-gamma (IFNꝩ) after stimulation. These stimuli included TLPDC or rhSPAG9 DCs (1:50 ratio of DCs/PBMCs) or PMA (10 ng) and ionomycin (100 ng) as the positive control. The production of IFNγ was analyzed after 72 h of incubation in the Cell Gro medium^® in the presence of 10 mg/mL of brefeldin A (Sigma, MA, USA). In all samples, incubation was performed at 37 °C in a 5% CO₂ incubator. The cells were washed, fixed, and subsequently stained for surface markers such as anti-CD4 PC5, anti-CD8 APC, and an intracellular marker anti-IFNꝩ FITC (all antibodies were IgG1, from Beckman Coulter Inc., CA, USA).