Bv605 rat anti mouse cd86 clone gl1

High-glucose Dulbecco’s modified Eagle’s medium (DMEM), phosphate-buffered saline (PBS) (pH 7.4) and trypsin-EDTA were sourced from the Laboratory of General Chemistry of the Institute of Immunology and Experimental Therapy, PAS (Wroclaw, Poland). Tris (hydroxymethyl) aminomethane (Tris), Sephacryl 100-S HR resin, bacterial lipopolysaccharide (LPS) from E. coli (serotype 055:B5), 3-(4,5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide (MTT) and Tween-20 were purchased from Sigma (St. Louis, MO, USA). L-glutamine and antibiotics (penicillin/streptomycin mixture) were purchased from BioWest (Nuaillé, France). Reagents for SDS-PAGE were purchased from Bio-Rad (Hercules, CA, USA). Molecular weight marker PageRuler™ Plus Prestained Protein Ladder, 10 to 250 kDa was purchased from Thermo-Scientific (Waltham, MA, USA). The U0126 inhibitor was obtained from Cell Signaling Technology (Leiden, The Netherlands), and SP600125 and LY294002 inhibitors were obtained from MedChemExpress (Monmouth Junction, NJ, USA). The antibodies used for flow cytometry analysis were: APC hamster anti-mouse CD80 (clone 16-10A1, BD Pharmingen, San Diego, USA) and BV605 rat anti-mouse CD86 (clone GL1, BD Horizon, Franklin Lakes, NJ, USA).

BMDM cells were seeded on 24-well flat-bottomed plate (3 × 10⁵/well) and cultured overnight in 10% FBS complete medium (37 °C, 5% CO₂/95% air). Next, the cells were stimulated with yolkin (100 µg/mL) or LPS (1 µg/mL) for 24 h. The next day, cells were stained with DAPI (Sigma) and antibodies for flow cytometry analysis: APC hamster anti-mouse CD80 (clone 16-10A1, BD Pharmingen) and BV605 rat anti-mouse CD86 (clone GL1, BD Horizon). Briefly, the staining procedure was as follows: cultured cells were harvested with warm PBS, washed twice (1300 rpm, 5 min) and stained with DAPI (10 min, RT, in the dark). Then, cells were washed with 1% FBS in PBS (1300 rpm, 5 min), suspended in 20% FBS and incubated for 30 min (4 °C, in the dark) to reduce the non-specific binding of antibodies. The cells were washed twice, as previously described, then suspended in 1% FBS in PBS and labelled with CD80 (1:100) and CD86 (1:100) antibodies (30 min, 4 °C, in the dark). Next, the cells were washed twice and suspended in 400 µL of 1% FBS in PBS and used for analysis.