Fitc latex beads

In this experiment PMA-differentiated macrophages from THP-1 and U937 cells in M0, M1 and M2 conditions at the end of the 48 h stimulation period were incubated for 30 min (at 37 °C and 5% CO₂) with 8 μL of 2.0 μm FITC-latex beads (L4530; Sigma-Aldrich, St Louis, MO, USA) added to 500 μL of complete culture medium. After the 30-min incubation, cells were thoroughly washed with PBS, enzymatically dissociated from the culture substrate (Accutase, BD Biosciences), resuspended in PBS + 2% FBS and analyzed by flow cytometry (BD FACS Verse, BD Biosciences). The experiment was performed in triplicate with acquisition of 10,000 events in each sample.

Medium, supplements, and fetal bovine serum (FBS) for cell culture were purchased from GIBCO BRL (Grand Island, N.Y.). Small molecule inhibiting agents: ALK5 inhibitor SB431542, PI3K inhibitor LY294002, NF-κB inhibitor BAY11-7082, ERK inhibitor PD98059 were obtained from Beyotime. Lipopolysaccharide (LPS), phorbol-12-myristate-13 acetate (PMA) and FITC-latex beads were purchased from Sigma-Aldrich (St Louis, MO). Primary antibodies against SNAIL, p-Akt (Ser473), Akt, p-Smad2, Smad2, p-Erk1/2, p-β-catenin, p-IκBα were purchased from Cell Signaling Technology (MA, USA) and anti-β-catenin, β-actin, α-Tubulin, GAPDH were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). FITC- or PE-conjugated antibodies for flow cytometry were supplied as follows: F4/80, CD80, CD86, from eBioscience (San Diego, CA); CCR7, CD206 from BD Biosciences (San Jose, CA). Lipofectamine 2000 was purchased from Invitrogen (Carlsbad, CA, USA). Recombinant protein human and mouse TGF-β1, human IFN-γ, and murine macrophage colony stimulating factor (M-CSF) were bought from Pepro Tech (Rocky Hill, NJ). PrimeScript^® RT reagent Kit and SYBR^® Premix Ex Taq™ were products of TaKaRa. E.Z.N.Z^® HP Total RNA Kit was bought from Omega Bio-Tek (Norcross, CA, USA).

Phagocytosis was examined by assessing the cellular uptake of 2.0 μm-sized FITC-latex beads (Sigma, US). Beads were incubated with cells at 37°C for 30 min. Cells were then washed with precooled PBS and analyzed via flow cytometry (FC, Beckman Coulter, US).