Anti mouse cd163 percp

RAW 264.7 cells were seeded in 24-well plates at a density 5 × 10⁵ cells/well in a 12-well plate. At 80–90% confluency, the cells were incubated with the ZnPc-EVs for 24 h in serum-free medium. As controls, cells were incubated with LPS/ IFN-γ (both at 0,1 µg/mL, as described above) for M1 polarization or 0,04 µg/mL IL-4 (Peprotech, lot# 021,749 J2418) for M2 polarization. Cells were then collected, washed in PBS and reconstituted in Fluorescence-Activated Cell Sorting (FACS) buffer (PBS with 0.5% Bovine Serum Albumin (BSA) and 0.02% sodium azide). The cells were then stained with Anti-mouse CD163- PerCP (eBioscience, San Diego, CA, USA) or anti-mouse CD68-FITC (eBioscience, San Diego, CA, USA) and analyzed by flow cytometry on an LSR II (BD Biosciences, San Jose, CA, USA).

Mouse anti-E-cadherin, mouse anti-vimentin, mouse anti-STAT3, mouse anti-NF-κB antibodies, and Hoechst were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Cer (from porcine brain) was purchased from Avanti Polar Lipids (Alabama, USA), PA was purchased from Sigma-Aldrich (Darmstadt, Germany), and anti-mouse CD163- PerCP, anti-mouse KI-67-APC, anti-mouse CD68-FITC, and anti-mouse IL-10-FITC were obtained from eBioscience (San Diego, CA, USA). Recombinant mouse IL-4, IFN-δ, and anti-IL-10R inhibitor were purchased from PeproTech (Rocky Hill, NJ, USA).
For immunofluorescence labeling, purified rabbit anti-Vimentin, rabbit anti NF-κB, rabbit anti-E-cadherin, and mouse anti-STAT3, and the secondary antibodies goat anti-mouse and goat anti-rabbit Alexa® Fluor 555 (Thermo Fisher Scientific) were used. Rabbit anti-β-actin (LI-COR Biosciences, Lincoln, Nebraska, USA) were used for immunoblotting.

Mouse anti-E-cadherin (Cat # MA1-06304) and mouse anti-vimentin (Cat # PA5-96191) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Anti-mouse CD163- PerCP (Cat # 46-1631-82) and anti-mouse CD68-FITC (Cat # 11-0689-42) were obtained from eBioscience (San Diego, CA, USA). Recombinant mouse IL-4 (Cat # 214-14) and IFN-γ (Cat # 315-05) were purchased from PeproTech (Rocky Hill, NJ, USA). Mitomycin (Cat # M5353) was purchased from Sigma-Aldrich (Amsterdam, The Netherlands).
For immunofluorescence labeling, purified rat anti-E-cadherin (Cat # 13-5700), the secondary antibodies goat anti-mouse (Cat # A32723), and goat anti-rat Alexa^® 488 (Catalog # A-11006, Thermo Fisher Scientific) were used. Rabbit anti-β-actin (Cat # 926-42210, LI-COR Biosciences, Lincoln, NE, USA), GRP94 (Cat # MA3-016, ThermoFisher), HSP70 (Cat # 4872S, Cell Signaling, Danvers, MA, USA), Flotillin-1 (Cat # 3244S, Cell Signaling), Integrin α6 (Cat # 3750S, Cell Signaling) and Integrin β1 (Cat # 4706S, Cell Signaling) were used for immunoblotting.