Enzyme free cell dissociation buffer

Tissue-culture-treated plates (12-well polystyrene plates, VWR, Radnor, PA, USA) were incubated with 5 µg/mL laminin-511-E8 solution (iMatrix-511) in sterile water for 3 h at room temperature. hiPS cells were dissociated from Matrigel-coated plates by treatment with enzyme-free cell dissociation buffer (Thermo Fisher Scientific, Waltham, MA, USA) and centrifuged (Avanti J15-R, Beckman-Coulter, Pasadena, CA, USA) at 200 g for 5 min in DMEM/F12. Then, the cells were resuspended in a mesoderm differentiation medium containing DMEM/F12 with GlutaMax (GIBCO) supplemented with 100 ng/mL activin A (Thermo Fisher Scientific, Waltham, MA, USA), 3 µM CHIR99021 (Stemgent, Cambridge, MA, USA), 10 µM Y27632 (TOCRIS, Minneapolis, MN, USA), and 1 × B27 serum-free supplement (GIBCO, Waltham, MA, USA), and 100,000 cells were plated onto each well of a 12-well plate coated with the laminin-511-E8 solution. After 2 days of differentiation, the cell culture medium was switched to an intermediate mesoderm induction medium containing DMEM/F12 with GlutaMax supplemented with 100 ng/mL BMP7 (Thermo Fisher Scientific, Waltham, MA, USA), 3 µM CHIR99021, and 1 × B27 serum-free supplement, and the cells were incubated for a minimum of 14 days with daily medium replenishment.

All studies requiring animals were carried out according to protocols
approved by the Institutional Animal Care and Use Committee (IACUC)
at the University of California, Irvine, which is fully accredited
by AAALAC. Bone marrow cells were flushed from femurs of 6–12
week old female C57BL/6J mice (Jackson Laboratories). The isolated
cells were treated with ACK lysing buffer to remove any red blood
cells (Thermo Fisher Scientific), and subsequently cultured in media
consisting of Dulbecco’s modified Eagle’s medium (DMEM)
(pH 7.4) supplemented with 10% heat-inactivated fetal bovine serum
(FBS), 2 mM l-glutamine, 1% penicillin/streptomycin (all
components from Thermo Fisher), and 10% conditioned media with macrophage
colony-stimulating factor (M-CSF) (macrophage culture media). The
cells were fed with the same media on day 3 and dissociated from the
culture plate on day 6 using an enzyme-free cell dissociation buffer
(Thermo Fisher Scientific). All of the experiments with the BMDMs
were performed using freshly differentiated cells.

HBSS (with Ca²⁺ and Mg²⁺), Dulbecco’s Modified Eagle’s Medium (DMEM; high glucose), 0.05% trypsin solution and penicillin/streptomycin solution, and enzyme-free cell dissociation buffer were purchased from ThermoFisher Scientific (Waltham, MA, USA). Fetal bovine serum (FBS) was obtained from Bodinco (Alkmaar, the Netherlands). Linear 25 kDa polyethylenimine (PEI) was purchased from Polysciences (Warrington, PA, USA). Bovine serum albumin (BSA) was obtained from Melford (Ipswich, UK). Furimazine (N1130) was purchased from Promega (Madison, WI, USA). The 96-well white and black cell culture plates used were obtained from Greiner Bio-one (Kremsmünster, Austria). White low-volume 384-well plates were bought from Corning (Corning, NY, USA). Human recombinant CXCL12, CXCL10, and the fluorescently labeled chemokines CXCL12-AZD488, CXCL12-AZD546, CXCL12-AZD594, CXCL12-AZD647, and CXCL10-AZD488 were purchased from Protein Foundry (Milwaukee, WI, USA). IT1t (Bio-Techne Ireland Limited, Dublin, Ireland), AMD3100 and Burixafor were purchased from MedChemExpress (Princeton, NJ, USA), and VUF25444, VUF15485, VUF25550 and VUF16545 were synthetized in-house.

One day following polarization of macrophages into M(-), M(IL-4), and M(HAGG+IL-1β), polarization media was washed off, and cells were treated with 20 ng/mL IFN-γ, 20 ng/mL IL-10, 1 μg/mL LPS (MD Biosciences), 1 μg/mL R848 (InvivoGen), 1000 U/mL IFN-α2a (PBL Assay Science), 10 μg/mL Poly (I:C) (InvivoGen), or 10 μg/mL ODN2395 CpG (InvivoGen) overnight. On the following day, macrophages were harvested using enzyme-free cell dissociation buffer (ThermoFisher).

Heparinized whole blood was obtained from normal healthy human donors (Biological Specialty Corporation) and informed consent was obtained from all donors. Biological Specialty Corporation obtains samples from FDA-registered collection centers and thus separate IRB approval is not required. CD14+ monocytes isolated from PBMC were used to generate monocyte-derived macrophages as previously described [11 (link)]. One day prior to the phagocytosis assay the monocyte-derived macrophages were either left untreated in M-CSF media (M(-) macrophages) or treated overnight with 20 ng/mL M-CSF and 300 ng/mL interferon-gamma (IFN-γ) (PeproTech) (M(IFN-γ)), 50 ng/mL IFN-γ and 50 ng/mL LPS (MD Biosciences) (M(IFN-γ+LPS)), 20 ng/mL IL-4 (PeproTech) (M(IL-4)), 20 ng/mL IL-1β (PeproTech) and 50 μg/mL heat aggregated IgG (HAGG) (M(HAGG+IL-1β)) or 20 ng/mL IL-10 (PeproTech) and 20 ng/mL TGFβ (PeproTech) (M (M(IL-10 + TGFβ)). Macrophages were harvested using Enzyme-Free Cell Dissociation Buffer (ThermoFisher).