Tnf a pacific blue

The following monoclonal antibodies reactive with human species were used for staining: CD8-VioGreen (BW135/80, cat.: 130-113-726, 1:50), CD4-APC-Vio770 (VIT4, cat.: 130-113-211, 1:50), CD154/CD40L-PE-Vio770 (5C8, cat.: 130-096-793, 1:10), CD20-VioGreen (LT20, cat.: 130-113-379, 1:50), CD14-VioGreen (TÜK4, 130-113-153, 1:50) (all from Miltenyi Biotec, Bergisch Gladbach, Germany);
IL-13-FITC (PVM13-1, cat.: 11-7139-42, 1:20 or 85BRD, cat.: 11-7136-42, 1:20), IL-4-PE (8D4-8, cat.: 12-7049-42, 1:20) (all from Thermo Fisher Scientific); TNFa-Pacific Blue (MAb11, cat.: 502920, 1:100), IFNg-PerCp-Cy5.5 (4S.B3, cat.: 502526, 1:20), CD3-APC (Okt3, cat.: 17-0037-42, 1:50) (all from Biolegend, San Diego, CA, US). Fixation and permeabilization were performed using the Foxp3 Transcription Factor Staining Buffer Set (Thermo Fisher Scientific) according to the manufacturer’s suggestions followed by intracellular cytokine and CD154/CD40-L staining. Fixable viability dye was used in eFluor-506 (cat.: 65-0866-14, 1:1000, Thermo Fisher Scientific).
Cells were acquired using BD FACSCanto II (with Diva software, Heidelberg, Germany) and post-acquisition data analysis was carried out using FlowJo software (TreeStar, Ashland, OR, US).