Human ab serum

To generate human peripheral blood-derived macrophages, buffy coat was isolated from leukapheresis cones by Ficoll-Paque centrifugation. Monocytes were enriched from human PBMC by CD14 beads positive selection (Miltenyi Biotec) and cultured in IMDM supplanted with 10% human AB serum (omega scientific, Cat# HS-20) for 7 days for them to differentiate to macrophages.
Differentiated macrophages were then replated to low-bind plate at 0.15 million cells per 24-well plate, in 500 µl medium overnight before stimulated with various condition. Human macrophage medium was used as M0 reference culture condition, while 10 ng/ml IFNg plus 10 ng/ml LPS supplement were used as M1 reference, and 10 ng/ml IL-10 supplement used as M2 reference. Cells were stimulated with 10 ng/mL chemerin9 for 72 hours, before stained with FITC anti-human CD11b (M1/70), PE anti-human HLA-DR (L243), BV510 anti-human CD86 (FUN-1), AF647 anti-human CD206 (15–2), and APC.Cy7 anti-human CD163 (GHI/61). Data were acquired on Cytek Aurora flowcytometry, analysis on Flowjo. MFI quantifications were normalized to M0 condition, and statistically analyzed by 2-way ANOVA, with Dunnett’s multiple comparisons test, all groups were pair-analyzed with M0 group as reference.

PBMCs were obtained by density gradient centrifugation using Ficoll-Hypaque (GE Healthcare, Chicago, IL, USA) and stored frozen until the testing of multiple timepoints to avoid interassay variability. Frozen PBMCs were thawed, resuspended, and plated into 24-well tissue culture plates (Corning, Corning, NY, USA) at 2–3 × 10⁶ viable cells/well in CTL media containing RPMI (Thermo Fisher Scientific, Waltham, MA, USA), 10% human AB serum (Omega Scientific, Inc., Tarzana, CA, USA), and 1% Pen/Strep-L-glutamine (Gibco, Thermo Fisher Scientific, Waltham, MA, USA). These PBMCs were stimulated with either the HER2 ECD or the HER2 ICD peptide mix (PM-ERB_ECD, PM-ERB_ICD; JPT Peptide Technologies GmbH, Germany) at 1 µg/ml in a 37°C humidified 5% CO₂ atmosphere in the presence of recombinant human IL-7 (5 ng/ml, Peprotech, Cranbury, NJ, USA) on day 0. A healthy donor control was stimulated in a similar way with 1 μg/ml CMVpp65 peptide (Mimotopes Pty Inc., Australia). Recombinant human IL-2 (Tecin; Hoffmann-La Roche, Switzerland) was added on day 3 at 12.5 units/ml. Cells were fed every 2–3 days by removing half of the culture supernatant and replacing it with fresh CTL media containing IL-7 and IL-2. The cells were harvested on days 10–12. If the cultures needed to be fed <48 h before the assay, the media were replaced without the addition of cytokines.