Ficoll plus gradient

Healthy donors were recruited from the Blood Donor Services of La Paz University Hospital. All participants provided written informed consent in accordance with the ethical guidelines of the 1975 Declaration of Helsinki and the Committee for Human Subjects of La Paz University Hospital (HULP: PI-3521) and the Clinical Research Ethics Committees of the Hospitals of Madrid (17.10.1125-GHM). Peripheral blood mononuclear cells (PBMCs) from healthy volunteers’ blood were isolated by Ficoll-Plus gradient (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA). Macrophages were obtained from PBMCs by a previously described adherence-positive selection protocol [31 (link)] and cultured in 6-well plates (Roche) with DMEM (Gibco-BRL Life Technologies) supplemented with 10% FBS (Gibco-BRL Life Technologies) and 0.01% penicillin/streptomycin (Thermofisher Scientific) at 37 °C in a humidified atmosphere with 5% CO₂.
After 15 days of macrophage differentiation, cells were either stimulated with IL-4 (10 ng/mL, PeproTech, PeproTech Ltd., London UK) or co-cultured with J82, UMUC1 or 5637 BC cell lines in a 1:5 tumor:macrophage ratio for 24 h. After that, cells were treated or not with 1 mM PBA for an additional 24 h. All the reagents used for the cell culture were endotoxin-free, as assayed with the Limulus amebocyte lysate test (Cambrex, Charles City, IA, USA).

The peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Plus gradient (GE Healthcare Bio-Sciences) as reported previously (4 (link), 13 (link)).

Peripheral blood was provided by the “Banc de Sang I Teixits” (Hospital Universitari de Bellvitge). Mononuclear cells were isolated from buffy coats using Ficoll-plus gradient (GE Healthcare Bio-Sciences). Neutrophils were isolated from the red fraction, then purified by dextran sedimentation. Purified cells were resuspended at 5 × 10⁶ cells/mL in RPMI supplemented with 10% of FBS and 50 U/mL streptomycin and penicillin. FACS analysis was performed to detect CD66b (G10F5, BD Bioscience) to confirm purity (98% average).
Neutrophil apoptosis and activation were analyzed culturing 10⁴ neutrophils per well in 96-well plates over 24 h in the indicated medium or CM. Apoptosis was measured using the Annexin AV Apoptosis Detection Kit (640930, BioLegend) and activation was detected by staining for CD11b following the previously described flow cytometry staining protocol.