Cd3 apc

PBMCs were isolated from blood samples of two patients, one with AML and another with CLL, using a Ficoll gradient (Axis Shield, Norway). Cells were collected in complete RPMI 1640 medium (pH 6.5). PBMCs (10⁶ cells) were then divided into five sets for each treatment and incubated with either ZIF-8 (5 μg·ml⁻¹), NV (10 μg·ml⁻¹), or NV-ZIF (5 μg·ml⁻¹) for 1 hour. Cells were then stimulated with PHA (100 ng·ml⁻¹) for 6 or 12 hours (the last 2 hours in the presence of brefeldin A). Intracellular cytokine staining was performed to determine the ability of CD8⁺ cells to express cytokines. The cells were surface stained with CD3⁺ APC (0.2 μg·μl⁻¹; R&D Systems, Minneapolis, MN, USA). They were then fixed in 4% paraformaldehyde, resuspended in 0.25% saponin (S4521; Sigma-Aldrich, Germany), and stained with anti–IFN-γ PE-Cy7 [PE-Cy7 mouse anti-human IFN-γ (BD Biosciences), 0.2 μg·μl⁻¹] and anti–TNF-α–PE-Cy7 [PE-Cy7 mouse anti-human TNF-α (BD Biosciences), 0.2 μg·μl⁻¹] antibodies. Samples were analyzed using a BD LSR II flow cytometer equipped with BD FACSDiva (BD Biosciences) software.