Apc conjugated anti tnfα

To measure cytokine expression, mouse N-9 cells were cultured with 5 µg/ml brefeldin A (eBioscience), which prevents protein transportation and leads to cytokine accumulation intracellularly. Cells were surface-stained with FITC-conjugated anti-CD11b (BioLegend, San Diego, CA) followed by intracellular staining of APC-conjugated anti-TNFα (eBioscience) and anti-IL-1β (eBioscience) with fixation and permeabilization buffer (eBioscience). The stained samples were analyzed on a Beckman Coulter CyAn ADP and data were analyzed with FlowJo software.

After metformin treatment, PBMCs were incubated at 2 × 10⁶ cells per well in RP10 media (RPMI, 10% Foetal Bovine Serum, 1% Penicillin–Streptomycin) alone or with 1X Cell Stimulation Cocktail (TONBO biosciences, cat.TNB-4975-UL100) for 4–6 h at 37℃. Cells were then harvested and intracellular stained according to the protocol of eBioscience Intracellular Fixation & Permeabilization Buffer Set (eBioscience, cat.88–8824-00). The following panel of mouse anti-human mAbs was used: APC-conjugated anti-IFN-γ (BD Pharmingen, 554702), APC-conjugated anti-TNF-α (eBioscience, 17–7349-82), PE-conjugated anti-Granzyme B (eBioscience, 12–8899-41), PE-conjugated anti-IL-6 (Biolegend, 501106). All samples were collected on FACSAria II BD (Mountain View, CA, USA). Data were analyzed using Flow Jo software (Tree Star, San Carlos, CA, USA).

PBMCs were thawed briefly in a 37°C water bath, washed in media (RPMI w/L-glutamine, 10% charcoal inactivated fetal bovine serum, 1% non-essential amino acids, and 1% penicillin/streptomyocin) and counted on a Beckman Coulter Cell Counter (Beckman Coulter, Brea, CA). 10^6 cells were plated per well and incubated at 37°C with 5% CO₂ for 2 hours prior to stimulation. Cells were treated with 2µM ODN2216 (Miltenyi Biotec, Bergisch Gladbach, Germany), 2µM ORN R2336 (Miltenyi Biotec, Bergisch Gladbach, Germany), 2µM ORN R2336 control (Miltenyi Biotec, Bergisch Gladbach, Germany) or media, and returned to incubator. After 2 hours, Golgi Plug (BD biosciences, San Jose, CA) was added to each well, and cells were subsequently incubated for another 4 hours until they were processed for flow cytometry staining as described above. The following antibodies were used in addition to those listed above and were all obtained from Miltenyi Biotec unless otherwise noted (Miltenyi Biotec, Bergisch Gladbach, Germany). PE-conjugated anti-IFNα, PE-conjugated anti-IL-6, PE-conjugated anti-human IgG1, APC-conjugated anti-TNFα, APC-conjugated anti-IL-10, APC-conjugated anti-human IgG1, APC-vio770-conjugated anti-HLA-DR, PerCP Cy5.5-conjugated anti-CD14 (ebioscience inc, Santa Clara, CA).