Percp cy5.5 conjugated anti human hla dr

PBMCs were isolated from peripheral blood of three grass pollen–allergic patients and three healthy controls as described above, and CD14⁺ monocytes were isolated by magnetic cell separation (MACS, with CD14⁺ beads, Miltenyi Biotec, Bergisch Gladbach, Germany) and analyzed by FACS (living CD14⁺ cells with a purity > 95%, BioLegend Zombie Aqua™ Fixable Viability Kit). For the generation of monocyte-derived dendritic cells (MoDCs) 3 × 10⁶ CD14⁺ monocytes were seeded into 6-well flat-bottom cell culture plates (Falcon, Thermo Fisher Scientific) in 3 mL T cell medium supplemented with 50 U/mL rhIL-4/rhGM-CSF (both from Miltenyi Biotec) and incubated at 37 °C and 5% CO₂ for 7 days. At day 4, the same amount of T cell medium supplemented with 50 U/mL rhIL-4/rhGM-CSF was added. After 7 days, immature MoDC (the purity of CD14⁻ MoDCs was > 99% (data not shown)) was analyzed by FACS analysis (APC-Cy7-conjugated anti-human CD80, PE-CF594-conjugated anti-human CD86, FITC-conjugated anti-human CD209, PerCP-Cy5.5-conjugated anti-human HLA-DR, and AF700-conjugated anti-human CD14, all from BD Biosciences, San Jose, CA, USA), and viability was checked by propidium iodide staining.

A total of 1 × 10⁵ cells/mL immature MoDCs were seeded into 96-well flat-bottom plates (Nunc, Thermo Fisher Scientific) and stimulated with 1 µg/mL lipopolysaccharides (LPS) or 1 µg/mL lipoteichoic acid (LTA) as positive control or 1 µg/mL GPE w/o 2 × 10⁵ CFU/mL Pollagen®, 1 µl Pollagen® supernatant, 2 × 10⁵ CFU/mL Kallergen®, or 10 µl Kallergen®. Untreated MoDCs were used as negative control. After 24 h of maturation at 37 °C and 5% CO₂, cytokine concentrations in culture supernatants were analyzed by LEGENDplex™ Human Inflammation Panel 1 (13-plex) assay (BioLegend) according to the manufacturer’s protocol. Furthermore, the expression of maturation markers CD80 (APC-Cy7-conjugated anti-human CD80, BD Biosciences), CD83 (APC-conjugated anti-human, BD Biosciences), CD86 (PE-CF594-conjugated anti-human CD86, BD Biosciences), CD209 (FITC-conjugated anti-human CD209, BD Biosciences), HLA-DR (PerCP-Cy5.5-conjugated anti-human HLA-DR, BD Biosciences), CD14 (AF700-conjugated anti-human CD14, BD Biosciences), and viability (Zombie NIR™ Fixable Viability Kit, BioLegend) was analyzed by flow cytometry.