αmem glutamax 1

Peripheral blood mononuclear cells (PBMCs) were either isolated from leukoreduction system chambers (LRSCs) from healthy donors or from blood samples of RA patients according to a published protocol (19 (link)). In total, 3 × 10⁵ cells were plated in 100 µl adherence medium [αMEM + GlutaMAX-I (Gibco) + 1% FCS/1% PS] per well of a flat-bottomed 96-well plate for 90 min (5.5% CO₂ and 37°C). Non-adherent cells were removed by flushing, and residual cells were cultured in 200 µl of OC medium [αMEM + GlutaMAX-I (Gibco) + 10% FCS/1% PS] supplemented with 30 ng/ml hu M-CSF (Peprotech), 10 ng/ml hu RANKL (Peprotech), and 1 ng/ml hu TGFβ (BioLegend) at 5.5% CO₂ and 37°C. The OC medium was replaced on days 3 and 6. Moreover, 25 µg sCD83 or the corresponding volume of PBS was added on days 0, 3, 4, 5, and 6. On day 7, fully differentiated osteoclasts were processed analogously to the murine protocol for TRAP staining and RNA extraction.

Total bone marrow cells were isolated from C57BL/6 mice (7 weeks) by flushing the femur and tibia. Blood cells were lysed using 0.8% ammonium chloride for 5 min at 37°C. The cells were plated overnight in OC medium (αMEM + GlutaMAX-I (Gibco) + 10% fetal calf serum (FCS)/1% PS) supplemented with 5 ng/ml M-CSF (Peprotech). Non-adherent bone marrow-derived monocytes (BMMs) were collected, washed, and further cultured in OC medium supplemented with 20 ng/ml M-CSF and 20 ng/ml RANKL (Peprotech) in 96-well plates at a density of 1 × 10⁶ cells/ml. The medium was changed on day 2. These cells were incubated daily with 25 μg/ml sCD83 from day 1 onwards. For experiments on the therapeutic potential of sCD83, 25 µg/ml sCD83 was only added on day 2 to the culture medium. At day 3, fully differentiated osteoclasts were washed with PBS and fixed with fixation buffer (25 ml citrate buffer + 65 ml acetone + 8 ml 37% PFA). Osteoclast differentiation into mature multinucleated TRAP⁺ cells (≥15 nuclei) was evaluated by TRAP staining using the leukocyte acid phosphatase kit 386A (Sigma-Aldrich) according to the manufacturer’s instructions. For RNA analyses, the cells were lysed in QIAGEN’s RLT PLUS buffer containing 1% β-mercaptoethanol.