Pe conjugated anti cd86

Approximately 1 × 10⁶ THP-1 cells were seeded in 6-well plates. After PMA stimulation, cells were incubated with PE-conjugated anti-CD86 (1:20, BioLegend) and APC-conjugated anti-CD206 (1:20, BioLegend) at 37 °C for 20 min in the dark. The fluorescence intensity was measured using a flow cytometer (Beckman).

Tissue culture reagents were purchased from Anprotec (Bruckberg, Germany), Biochrom GmbH (Berlin, Germany), PAN biotech (Aidenbach, Germany), Thermo Scientific (Langenselbold, Germany), Merck and Sigma. Antibodies against phosphorylated NF-kB, AMPKα (Thr172), 4E-BP1 (Thr37/46), p70 S6 Kinase (Thr 389), Tyr-100, IKKα/β and total NFATc1 as well as RelB were purchased from Cell Signaling Technology (Leiden, The Netherlands). Antibodies against GAPDH, β-actin and cytochrome C were obtained from Proteintech (Manchester, UK). OXPHOS antibody cocktail was purchased from abcam (Cambridge, UK). Secondary horseradish peroxidase (HRP)-linked antibodies were obtained from Cell Signaling Technology (anti-rabbit IgG, anti-Mouse IgG). PE-conjugated anti-F4/80, PE-conjugated anti-CD86, PE-conjugated CD-115, CD265 (RANK), and PE- conjugated IgG2a were provided by BioLegend (San Diego, CA, USA). PE-conjugated anti-CD80 was purchased from BD Biosciences (Heidelberg, Germany). SYTOX Green was procured from Thermo Scientific (Langenselbold, Germany), and MitoTracker DEEP Red FM stain from Cell Signaling Technology (Leiden, The Netherlands). PCR primers were purchased from Apara (Denzlingen, Germany) or Biomers (Ulm, Germany).