Tnf α af647

BAL cells and PBMCs were isolated and stimulated at 1 × 10⁶ cells/mL with Ag85A, ESAT-6/CFP-10 peptides (2 µg/mL each), and PPD (20 µg/mL); unstimulated cells and SEB-stimulated cells were used as negative and positive controls, respectively. Brefeldin A (Sigma) was added to the cells 2 h after stimulation, and cells were incubated overnight at 37 °C and 5% CO₂.
Harvested BAL cells and PBMCs were stained with the live/dead red viability marker (Thermo Fisher) for the exclusion of dead cells, followed by surface staining with CD4-Pacific Blue (Biolegend, San Diego, CA, USA), CD14, and CD19 on ECD (Beckman Coulter, Brea, CA, USA). Cells were then permeabilised and stained with CD3-AF700 (Ebioscience, San Diego, CA, USA), CD8-APC/H7 (BD), IFN-γ-PECY7 (Ebioscience) and TNF-α-AF-647 (Biolegend), IL-2 PE (Beckman Coulter), and IL-17-AF488 (Biolegend).
Cytokine-producing CD4+ and CD8+ T-cells were gated on CD3+, CD14−, CD19− single T cells. Data were analysed using Flowjo (BD) and are presented as background-subtracted antigen-specific responses.

Intracellular cytokine staining was performed as previously described(1) using the following markers: CD3-APCH7, CD107a PECy7, CD14-Pacific Blue, CD16-Pacific Blue, CD4-PECy5.5, IFN-γ-PerCPCy5.5, CD45RO-AF700, CD19-Pacific Blue (BD Biosciences, San Jose, CA), TNFα-AF647, CD8-BV570, CCR7-BV711 (BioLegend, San Diego, CA), granzyme B-PE Texas Red (Invitrogen), CD27-PECy5 (eBioscience, San Diego, CA) perforin-FITC(Abcam, Cambridge, UK). Prepared cells were acquired using an LSR II flow cytometer equipped with BD FACSDiva software (BD Biosciences). Acquired data was analyzed using the FlowJo software version 7.6.3 (Tree Star).