Cd40 apc

Cells were harvested for staining and incubated with Fc-block for 10 min at room temperature. Cells were stained for 1 h at 4°C with CD11c—PE or FITC, CD40—APC, CD80—FITC, CD86—APC-cy7 (BD bioscience). Cells were fixed using Fix & Perm (Life Technologies) following manufacturer's instructions. Intracellular staining was performed using iNOS—PE (ebioscience). For glucose uptake assays, 50 ul fluorescent glucose analog (2-NBDG-FITC) was added to the cells 120 min before harvest. CFSE labeled T. gondii was used for uptake assays. A total of 30, 000–50, 000 events per sample was acquired on a BD FASCanto and data analysis was carried out using FlowJo software.

A total of 2.5 × 10⁶ liver-isolated APCs from different groups were incubated with the same amount of E. coli colony-forming units (CFUs) in the presence of autologous rat serum with HBSS in a final volume of 1 mL in an orbital shaker at 37 °C at 50 rpm. APCs and E. coli were incubated together for 20 min to measure baseline E. coli-binding and internalizing capacity of APCs (T₀) and 2 h (T₂) to measure APCs’ ability to kill E. coli. Cells were washed after incubations and subjected to a 30% sucrose centrifugation to separate non-bound E. coli from APCs. Resting APCs were resuspended in 1 mL HBSS 5% serum and diluted 1:5 in sterile water to release APC content. Dilutions were seeded in agar plates (Biomerieux, Marcy l’Etoile, France) and incubated overnight at 37 °C. CFUs were counted, and values were represented for each APC in the different study groups.
Cell surface expression of CD40-APC, CD80-PE, and CD86-FITC and their respective isotype controls (BD Biosciences, San Diego, CA, USA) was measured by flow cytometry in isolated DCs, HMs, and LSECs from animals in the different study conditions using a FACSCanto II flow cytometer operated by FACSDiva software (BD Biosciences, San Diego, CA, USA). Representative images of CD40, CD80, and CD86 and their isotype-matched controls are shown in Figure S4 for control rats.

BMDC were stained with 1 µM CFSE for 10 min at 37 °C in PBS, washed and seeded at 5 × 10⁴ cells/well in 96 well suspension plates. Next, BMDC were co-cultured with MCA205 cells in ratios 1:1; 1:5; 1:10 (BMDC:MCA205) or stimulated with 250 ng/ml of LPS (#L-2630, Sigma Aldrich). After O/N co-culture, cells were collected, washed in ice-cold FACS buffer and stained for surface markers: CD11c-BV650 (#117339, BioLegend, 1:200), MHCII-APC/eFluor700 (#47-5321-82, eBioscience, 1:100), CD86-PE/Cy7 (#105116, BioLegend, 1:200), CD80-PE/Cy5 (#104712, BioLegend, 1:200), CD40-APC (#558695, BD Pharmingen, 1:200) and CD274-PE (#12-5982-82, eBioscience, 1:200). The acquisition was performed with a LSRII flow cytometer using BD FACSDIVA 8.0 software and the analysis was performed using FlowJo 10.2 software.