Tnf α alexa700

To analyze the production of cytokines by PBMCs, fresh PBMCs were cultured in ultralow attachment plates (Corning Incorporated, Baltimore, MD, USA) (1 mL of cells at 10⁶ cells/mL) and incubated for 4 h at 37 °C with 5% CO₂. PBMC stimulation was performed by adding LPS (5 µg/mL, Sigma-Aldrich Chemistry, Madrid, Spain) and monensin (50 µg/mL, Sigma). Next, the cells were labeled with CD14-PerCP and CD16-Alexa647 (Becton Dickinson) MoAbs and the vital dye Aqua-QD565. For intracytoplasmic staining, the cells were fixed and permeabilized (Fix and Perm, Caltag Laboratories, Burlingame, CA, USA), and cytokines were stained with IL-1β-FITC, IL-10-PE, IL-6-V505, and TNF-α-Alexa700 (Becton Dickinson) MoAbs.

The proportions of monocyte subsets were determined in fresh PBMCs by seven-color polychromatic flow cytometry in a FACSAria II cytometer using FACSDiva software (Becton Dickinson, NJ, USA). To analyze the production of cytokines by PBMCs, 1 million of fresh PBMCs were cultured in ultralow attachment plates (Corning Incorporated, ME, USA) (1 ml of cells at 10⁶ cells/ml) and incubated for 4 h at 37°C with 5% CO₂. PBMC stimulation was performed by adding LPS (10 µg/ml, Sigma-Aldrich Chemistry, Spain) and monensin (50 µg/ml, Sigma). Next, the cells were labeled with CD14-PerCP and CD16-Alexa647 (Becton Dickinson) MoAbs and the vital dye Aqua-QD565. For intracytoplasmic staining, the cells were fixed and permeabilized (Fix and Perm, Caltag Laboratories), and cytokines were stained with IL-1β-FITC, IL-10-PE, IL-6-V505 and TNF-α-Alexa700 (Becton Dickinson) MoAbs.

T cells were magnetically purified from PBMC using a PAN T-cell isolation kit (Miltenyi Biotec) from healthy donors. Healthy donor T cells (1 × 10⁵) were then cultured with bead-purified (Miltenyi Biotec) CD19+ B cells from CLL patients (1 × 10⁶) at an optimal 1:10 ratio per well in the presence or absence of CXCL12 (250 ng/ml), lenalidomide (10 µM), CXCR4, or mouse anti-human IL-10 blocking antibodies (2 µg/ml, R&D). Each experiment was performed in duplicate. After 48 h of culture, T cells were magnetically purified again as described above (purity >95%) and activated with anti-CD3/CD28 magnetic Dynabeads (Invitrogen) for 6 h. A negative control (no stimulation) was included in every experiment. Brefeldin A (10 µg/mL) and CD107a PE-CF594 (BD Biosciences) were added to the culture. Cells were stained with a Live/Dead-Aqua (Invitrogen), CD3-BV650, CD8-FITC, CD4-APC-Cy7, fixed/permeabilized (BD Biosciences) followed by intracellular staining with IFN-γ-V450, TNF-α-Alexa 700, and IL-2-PE (BD Biosciences).