Fitc anti human cd80 antibody

Fresh PBMCs from two healthy donors were separated from whole blood samples using Ficoll-Paque^TM. CD14+ monocytes were selected using MACS human CD14 MicroBeads (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany), pre-separation filters (Miltenyi), and LS columns (Miltenyi) following the manufacturer’s recommendations. To induce M0 macrophages, isolated CD14+ monocytes were seeded onto FBS-coated 24-well plates at a density of 1 × 10⁵ cells/cm². Seeded cells were cultured for 7 days in RPMI 1640 media supplemented with 20% FBS and 100 ng/ml M-CSF (BioLegend, San Diego, CA, USA). On day 7, M-CSF-containing medium was removed, and appropriate stimulating media containing 100 ng/ml LPS (Sigma-Aldrich, St. Louis, MO, USA) and 20 ng/ml IFN-γ (BioLegend) for M1 induction or 20 ng/ml IL-4 (BioLegend) and 20 ng/ml IL-10 (BioLegend) for M2 induction were supplied. After an additional 48 h of culture, cells were collected by gentle scraping and then taken for fluorescence-activated cell sorting (FACS) analysis or scRNA-seq. The morphological changes that occurred during differentiation were observed every 2 days under a microscope. A fraction of the cells was triple-stained with PE/Cy7 anti-human CD14 antibody (BioLegend), FITC anti-human CD80 antibody (BioLegend), and PE anti-human CD163 antibody (BioLegend) and analyzed by FACSVerse and FACSuite v1.2 (BD Biosciences).

We harvested the cells 24 hours after LPS administration. The cells were stained with an FITC-anti-human CD80 antibody (BioLegend, San Diego, California, USA) or FITC mouse IgG1, k isotype (Becton, Dickinson and Company, Franklin Lakes, New Jersey, USA) for 30 minutes at 4°C after blocking the nonspecific Fc receptor using FC blocking reagent human (Miltenyi Biotec) for 10 minutes. After staining, the cells were washed immediately and resuspended in AutoMACS Runnig Buffer. Fluorescence data were collected using an Attune NxT Flow Cytometer, and the FITC-positive cell population was evaluated using an excitation wavelength of 488 nm. The flow cytometry files were analyzed using FlowJo software (Becton, Dickinson and Company).