Telaval 31 light microscope

For induction of M2-polarized TAMs, RAW 264.7 cells (5 × 105 cells/well in a 12-well plate) were cultured in complete medium with 10% FBS, supplemented with 20 ng/mL IL-4 for 24 h. After 24 h, RAW 264.7 cells were washed three times with serum-free (sf) medium and cultured for another 48hrs in sf medium supplemented with IL-4. After 48 h of serum starvation, RAW 264.7 cells were collected and used as M2-polarized TAMs. During the polarization process, RAW 264.7 supernatant was collected and used as M2-polarized TAMs-conditioned medium (CM). For induction of M1-polarized TAMs, RAW 264.7 cells (5 × 105 cells/well in a 12-well plate) were cultured in medium with 10% FBS, supplemented with LPS and IFN-γ for 24 h. After 24 h, RAW 264.7 cells were washed three times with sf medium and cultured for another 48 hrs in sf medium [37 (link)]. To assess the effect of PA and Cer on the polarization state of M2 macrophages, RAW 264.7 cells were first cultured for 24 h in the presence of IL-4 and 10% serum, as described above. After 24 h, the cells were washed three times with sf medium and resuspended in sf medium, substituted with 10 µM Cer or 10 µM PA. The cells were cultured for another 48 h in the presence of lipids. Cells morphology, as an indication of M2 polarization, was analyzed using a Telaval 31 light microscope (ZEISS, Oberkochen, Germany).

CT-26 cells (1 × 10⁴ cells/well) were grown in DMEM containing 10 % FBS in 12-well plates (BD Bioscience). In DMEM with 10 % FBS, M2-polarized TAMs (5 × 104 cells/insert) were seeded into the upper chamber of a transwell insert with an 8 μm pore size (Corning Inc., Corning, NY, USA). The next day, the M2-polarized TAMs were treated with 2.5 μg/ml NP3 (RA)-CHO in the presence or absence of 1.2 μg/ml anti-IL-10R inhibitor, and the cells were cultivated for another 48 hours at 37°C in a humidified incubator with 5% CO2. A Telaval 31 light microscope (ZEISS) was used to examine the morphology of the cells. CT-26 cells were collected after treatment and fixed with 2% PFA and permeabilized with 0.1 % Triton X-100. Next, the cells were incubated with mouse anti-Ki-67-APC (1:100) or mouse anti-IL-10-FITC (1:100) at 4°C for 60 minutes and, subsequently, analysed using flow cytometry [11] (link).

CT-26 cells (1 × 10⁴ cells/well) were seeded in 12-well plates (BD Bioscience) in DMEM medium supplemented with 10% FBS. M2-polarized TAMs (5 × 10⁴ cells/insert) were seeded into the upper chamber of the transwell insert with an 8 μm pore size (Corning Inc., Corning, NY, USA) in DMEM with 10% FBS. The following day, the culture inserts with M2-polarized TAMs were treated with 10 µM Cer or 10 µM PA, in the absence or presence of 1.2 µg/mL anti-IL-10R inhibitor, and cells were cultured for another 48 h in a humidified incubator with 5% CO₂ at a temperature of 37 °C. Cells were morphologically analyzed using a Telaval 31 light microscope (ZEISS). After treatment, CT-26 cells were collected and fixed with 2% PFA, permeabilized using 0.1% Triton, followed by incubation with anti-mouse KI-67-APC (1:100) or anti-mouse IL-10-FITC (1:100) at 4 °C for 60 min, and analyzed by flow cytometry. Moreover, CT-26 cells were collected for RNA extraction with TRIzol. The cell culture supernatants were collected and analyzed by ELISA.