Pam3csk4 pam

2 × 10⁵ BMMC were cultured in 0.25 mL of complete medium with or without VPA at 2 mM for 18 h. Cells were washed with HBSS and activated with different stimuli: L.m MOI 100:1 or Staphylococcus aureus Peptidoglycan (PGN; Sigma-Aldrich, USA) at 10 μg/mL or Pam3CSK4 (Pam; InvivoGen, USA) at 10 μg/mL, or Recombinant Listeriolysin-O (LLO; RayBiotech, USA) at 1000 ng/mL for 24 h. Supernatants were collected for the detection of TNF, IL-6, CCL2 (BioLegend, San Diego, CA., USA) and IL-13 (eBioscience, USA) by ELISA according to manufacturer’s instructions.

γδ T cells were purified from the pooled spleens of 3–5 mice by using a TCRγ/δ⁺ T Cell Isolation Kit according to the manufacturer's instructions (Miltenyi Biotec, Auburn, CA). The purity of γδ T cells was examined by staining with streptavidin-PE and anti-CD3 FITC. γδ T cells (1×10⁵ cells/well) were cultured for 2 days at 37°C in RPMI-1640 medium (Invitrogen, Carlsbad, CA) in 96-well plates coated with 5 µg/ml anti-CD3 (eBioscience, San Diego, CA) in the presence of 1 µg/ml of Pam3CSK4 (PAM, Invivogen, San Diego, CA), or 10 µg/ml of lipopolysaccharide (LPS, Sigma, St. Louis, MO) or 1 µg/ml of CL097 (Invivogen). At 48 h post-treatment, cells were harvested and stained for cell surface markers. Culture supernatant was harvested for measurement of cytokine production.