Recombinant human tgf β1

Protocols involving human tissues were approved by the human studies Internal Review Board at the University of South Carolina. Surgical skin samples were obtained from the Cooperative Human Tissue Network of the National Cancer Institute. Skin MCs were isolated and cultured as described previously (21 (link)) and were used after 6–12 weeks. Mast cell purity was determined to be 100% by toluidine blue staining. When applicable, human mast cells were sensitized 24 hour prior to the antigen (Ag) stimulation with the addition of 1 μg/ml DNP-specific mouse IgE (a gift from Dr. Daniel Conrad, VCU), washed to remove excess unbound IgE, and stimulated with 50 ng/ml DNP-HSA (Ag), for 16 hours. Where indicated, recombinant human TGFβ1 (10 ng/ml, BioLegend) was applied for 3 days prior to Ag stimulation and recombinant human IL-33 (100 ng/ml, BioLegend) was added at the same time as Ag. All supernatants were collected after 16 hours of stimulation. Each experimental condition was performed in triplicate determinations from 5 different donors.

Tissue culture-treated plates were first coated with 5 μg/mL anti-mouse CD3ε antibody (100314, BioLegend, San Diego, CA), at 37°C for 4 hr. Lymphocytes were harvested from mouse spleen through a 70 μm cell strainer as described above and resuspended in PBS. Then, these CD4+ T-cells were negatively isolated from splenocytes as described above. The isolated CD4+ T cells were resuspended into culture media (RPMI 1640 (11875093, ThermoFisher Scientific Inc., Waltham, MA) + 10% FBS (26140079, ThermoFisher Scientific Inc., Waltham, MA) + 10 mM HEPES + 1X Antibiotic-Antimycotic (15240062, ThermoFisher Scientific Inc., Waltham, MA) supplemented with 5 μg/ml anti-mouse CD28 antibody (102112, BioLegend, San Diego, CA), 50 ng/ml recombinant mouse IL-6 (575704, BioLegend, San Diego, CA), 5 ng/ml recombinant human TGF-β1 (580702, BioLegend, San Diego, CA), 10 ng/mL recombinant mouse IL-23 (589002, BioLegend, San Diego, CA), 10 μg/mL anti-mouse IL-4 antibody (504108, BioLegend, San Diego, CA), and 10 μg/mL anti-mouse IFN-γ antibody (505812, BioLegend, San Diego, CA) to a cell density of 0.8 - 1 x 10⁶/mL and incubated at 37°C with 5% CO₂ for 2 days. Fresh medium with the same supplements was added into each well to increase the initial volume by 50% and incubation was continued for another 2 days.

HL-60 cells (Sigma-Aldrich, St. Louis, MO) were cultured and differentiated as described in the Online Supplement. Primary human neutrophils were isolated from healthy donor blood using the EasySep Direct Human Neutrophil Isolation Kit (Stemcell Technologies, Vancouver, BC) per the manufacturer’s instructions. Neutrophil purity was confirmed with Kwik-Diff (Thermo Shandon) staining. Cells were cultured at 37°C, 5% CO₂ in Roswell Park Memorial Institute-1640 media with L-glutamine supplemented with 10% heat-inactivated Fetal Calf Serum, 1% penicillin/streptomycin, 50 mg/ml gentamicin and 5ng/ml recombinant human TGF-β1 (BioLegend) for 16 hours. Recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) (Sigma) was added at 100ng/ml as indicated for 30 minutes prior to harvesting.