Containing brefeldin a

For intracellular cytokine labelling, splenocyte suspensions (2*10⁵ cells/well) of young and aged mice were cultured with phorbol 12-myristate 13-acetate (PMA) (0.05 μg/mL) and ionomycin (0.5 μg/mL) (Sigma Aldrich) in medium for four hours in total. After one hour of culturing, GolgiPlug (containing Brefeldin A; 1:1000, BD Biosciences) was added to allow intracellular accumulation of produced cytokines. After labelling with anti-CD4, anti-CD8, and Live/Dead Fixable Aqua, cells were fixed and permeabilised and labelled intracellularly with anti-CD3 and for the presence of IFN-γ, TNF-α, IL-4, and IL-5 as described in our flow cytometric labelling protocol.

To determine whether the small compounds could stimulate lymphocyte activation, Peripheral blood mononuclear cells (PBMCs) activation assay was performed as previously reported [31 (link), 32 (link)]. Briefly, the PBMCs were isolated from blood samples of healthy donors by the Ficoll-Paque density gradient centrifugation, and re-suspended in an IMDM medium containing 10% FBS. PBMCs were seeded into round bottom 48-well plates at a density of 4 × 10⁵ cells/well, and stimulated with stimulatory antibodies of 1 μg/mL human anti-CD3 (clone: OKT3, eBioscience) and 0.5 μg/mL human anti-CD28 (clone: CD28.2, eBioscience). Indicated compound (100 μM) or medium with 1% DMSO as negative control were tested for the ability to enhance the activation of lymphocytes in PBMCs. Three days later, an intracellular cytokine staining assay was performed. Cells were incubated with protein transport inhibitor (Containing Brefeldin A) (BD Biosciences) for 4 h, and stained with antibodies of human anti-CD4 PerCP-Cy5.5 (clone: OKT4, eBioscience) and human anti-CD8 APC (clone: SK1, eBioscience) on ice for 30 min. Then, cells were fixed and permeabilized each for 30 min with the Foxp3/Transcription Factor Staining Buffer Set (eBioscience) according to the manufacture. Permeabilized cells were then stained with human anti-IFN-γ PE antibody (clone: 4S.B3, eBioscience) or an isotype control.

Thawed, washed, and rested lymphocytes were cultured in 96-well U-bottom plates, at a concentration of 2 × 10⁵ cells in a total volume of 200 µl of complete medium, for 18 h (37°C, 5% CO₂) with or without IL-12 plus IL-18 [1 ng/ml recombinant mouse (rm)-IL-12 (PeproTech, Rocky Hill, NJ, USA) plus 20 ng/ml rmIL-18 (MBL, Woburn, MA, USA)] or IL-2 [100 ng/ml rmIL-2 (PeproTech)]. Two microliters of anti-CD107a-FITC were added to each well after 14 h and GolgiPlug (containing Brefeldin A, 1/1,000 final concentration; BD biosciences, Oxford, UK) and GolgiStop (containing Monensin, 1/1,500 concentration; BD biosciences, Oxford, UK) were added after 15 h.