Il 6 apc

After digestion with collagenase II (2 µg/mL) for 45 min at 37 °C, the same-weight muscle samples were filtered by a 220 µm filter, washed with phosphate-buffered saline (PBS), centrifuged at 2500 rpm for 5 min, and then transferred to a 96-well plate. The antibodies used are as follows, CD45-PE, CD3-A780, NK1.1-APC, TNFα-PE, IL6-APC (Biolegend, U.S.A).
Flow cytometry was performed using a BD Biosciences LSR Fortessa flow cytometer, and the data were analyzed using FlowJo 10.

All cell culture supplies were obtained from Invitrogen (ThermoFisher Scientific, Waltham, MA), unless otherwise specified. Fetal bovine serum (FBS) was purchased from Atlanta Biologicals (Flowery Branch, GA). All TLR7/8 compounds were synthesized and characterized as previously reported^{27 (link)}. Frozen human PBMCs were purchased from Cellular Technology Limited (Shaker Heights, OH). Fluorophore labelled anti-human monoclonal antibodies (CD3-FITC, CD56-PE/Cy7, CD11c-PE/Cy7, CD19-PE, GranzymeB-PE, IL-6-APC, TNF-α-APC/Cy7, IFNγ-BV421 and IL-2-BV605) and Brefeldin A were purchased from Biolegend (San Diego, CA).

Peripheral blood mononuclear cells were incubated overnight with 10 ng/ml LPS (Sigma-Aldrich) in the presence of GolgiPlug (1:1,000, Becton Dickinson) after pre-stimulation with IFN-γ for 2 h. The cells were then incubated with conjugated primary antibodies in phosphate-buffered saline containing 0.5% bovine serum albumine for 30 min. The antibodies used were CD3-PE, CD14-Pacfic Blue, CD16-PE-Cy7, CD20-PE, and CD56-PE (all Biolegend) at 4°C and were incubated with EDTA for 15 min followed by incubation with FACS permeabilizing solution 2 (BD Biosciences) for 15 min. Next, conjugated antibodies to TNF-α-Percp-Cy5.5, IFN-γ-APC-Cy7, IL-1β-FITC, IL-6-APC, and IL-10-APC and their respective isotype controls (all Biolegend) were added to determine intracellular cytokine production. The cells were washed and analyzed using flow cytometry (FACSCanto II, BD Biosciences) and FACSDiva software (16 (link)).