Rh m csf

Human peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats using density gradient centrifugation on Ficoll-Paque according to manufacturer protocol. Monocytes were positively isolated using magnetic CD14 microbeads (Miltenyi, #130-050-201) and were cultured in a low-attachment flask for 5 days in RPMI (Gibco, #72400) supplemented with heat-inactivated FBS (Gibco, #F8067), Pen/Strep (Gibco, #15140) and [100 ng/mL] rh M-CSF (ImmunoTools, #11343115) to differentiate towards macrophages.

During ECM polymerization, HIO were retrieved from Matrigel droplets and made single cells using TryplExpress (Gibco, #12,605) and mechanical dissociation as previously reported^{15 (link),28 (link)}. 100,000 HIO cells were seeded in a 2 µL volume in the upper channel of the OrganoPlate and left to adhere against the gel for 45 min. After incubation, 50 µL of HISC medium devoid of A83-01 (attachment medium) was added to the upper inlet and the plates were returned to the incubator at a 70° angle for 4–6 supplementary hours. After 4–6 h, 50 µL of attachment medium was added to all remaining inlets and outlets and the plates were placed back in the incubator without medium perfusion for 24 h. On Day 1, plates were placed on an interval rocker (Perfusion Rocker Mini, Mimetas) switching between a + 7° and − 7° inclination every 8 min (37 °C, 5% CO₂) leading to a 121.2 µl/hr perfusion rate^{59 (link)}. Attachment medium was replaced with HISC medium two days after seeding, and HISC medium was refreshed every three days after that. During all experimentation, the lower channel medium was supplemented with [100 ng/mL] rh M-CSF (ImmunoTools, #11,343,115) to maintain the macrophages differentiated.

Purified monocyte subsets and NK cells were pre-incubated at 37 °C for 5 hrs with 100 ng/ml LPS (E. coli 0111:B4; Sigma Aldrich), 10 μg/ml R848 (Invivogen), 100 ng/ml rhIL-12 (Immunotools), 100 ng/ml rhIL-15 (R&D Systems), 20 ng/ml rhIFN-γ (Immunotools), 8 μg/ml S100A9 (Origene Technologies) or 100 ng/ml HMGB1 (R&D Systems) before they were used for ADCC assay with BATDA-labeled GM2-coated A549 as target cells at an E:T ratio of 10:1. Freshly isolated CD16− monocytes were cultured in complete IMDM either in the absence or presence of 50 ng/ml rhM-CSF (Immunotools), 50 ng/ml rhTGF-β (R&D Systems) or 50 ng/ml rhIL-10 (Immunotools) at 37 °C for 24 hrs before their ADCC activity was assessed on trastuzumab-coated SKBR3 at a 10:1 E:T ratio.