Anti cd80

Expression of CD90, CD105, CD45, and CD11b/c (eBioscience, USA) on MSCs surface was assessed by flow cytometry (Becton Dickinson, USA). The expression of CD11b/c, CD80, CD83, CD86, and MHC II (eBioscience, USA) on DCs surface was identified using flow cytometry. Each group of DCs were collected on the sixth day. After washing with PBS, the cells were immunolabeled with monoclonal anti-CD80, anti-CD83, anti-CD86, and anti-MHC II (eBioscience, USA) antibodies, as well as their isotype control antibodies. After that they were incubated in darkness at 4°C for 30 min and were detected using FACSCalibur flow cytometer.

Immature DCs (10⁷) were stimulated for 6 h with LPS (1 µg/ml E. coli strain O111:B4, Calbiotech Merck) or LPS in combination with IFN-γ (0.02 µg/ml, BD Pharmingen) with or without Dex (10⁻⁸ M, Sigma-Aldrich) for 20 min before adding other reagents. To monitor maturation, DCs (10⁵) were stained with 5 µl of mix including anti-MHC-I, anti-MHC-II, anti-CD11b, anti-CD11c, anti-CD80, anti-CD83 and anti-CD86 (all from eBioscience, Austria).
T-cells were stained with anti-CD3, anti-CD4, anti-CD8, anti-CD25 and anti-Vα2 TCR for OT-I and OT-II mice (eBioscience). Cell viability was analysed with DAPI (Sigma-Aldrich). Apoptosis was measured with Annexin V (BD Pharmingen). CFSE (7 µM, Invitrogen) or Cell Proliferation Dye eFluor 670 (CPD, 5 µM, eBioscience) were used to detect proliferation. Flow cytometry was done on an LSR II (BD Pharmingen). Data were analysed by FlowJo (Version 9.6.2 Treestar). The difference in apoptosis induction was calculated using absolute cell number, determined with BD Trucount tubes.
This method is based on lyophilized pellet, containing a known number of fluorescent beads, which dissolves once the monoclonal antibody reagent is added. Absolute numbers (cells/µl) of positive cells in the sample are calculated following the equation: number of cell events/number of bead events x Trucount bead concentration.

For the surface marker staining, cells were stained according to the manufacturer's directions. The following antibodies were used: anti-F4/80 (eBioscience), anti-CD80 (eBioscience, Waltham, MA, USA), anti-CD86 (eBioscience), anti-MHCII (eBioscience). To detect LC3II in splenic macrophages, Flowcellect autophagy LC3 antibody-based assay kit (Merk Millipore) was used according to the manufacturer's directions. To assess TLR7, Notch1 expression in macrophages, spleen cells were then stained with anti-F4/80 and resuspended with fixation/permeabilization solution (eBioscience) then stained with anti-TLR7 (IMGENEX, CO, USA), anti-Noch1 (eBioscience), respectively. Similarly, fixed and permeabilized cells were stained with anti-Hes-1 (abcam) or anti-P62 (Merk Millipore), and then stained with Alexa Fluor 647 conjugated goat anti-rabbit IgG secondary antibody (abcam) and Alexa Fluor 488 conjugated goat anti-mouse IgG secondary antibody (abcam), respectively. Annexin V-FITC and PI Apoptosis kit (eBiosciences) was used to examin the mortality of macrophages in vivo and in vitro. All of the flow cytometry data were aquired with the BD FACS Calibur cytometer and analyzed by FlowJo software.

Splenocytes and liver mononuclear cells were isolated as described previously [16 (link)]. Flow cytometric analysis was performed using BD FACSCalibur and FACSAria III instruments. Antibodies used in this study included fluorescein isothiocyanate (FITC)-labeled anti-mouse CD49b (DX5), anti-NK1.1, anti-CD4, and anti-CD11c; Phycoerythrin (PE)-labeled anti-mouse-CD69, anti-CD107a, anti-CD44, anti-CD86, and anti-IFN-γ; PE-cyanine 5.5-labeled anti-mouse CD3e and anti-CD8; and allophycocyanin (APC)-labeled anti-mouse CD314 (NKG2D), anti-CD69, anti-CD25, anti-CD80, anti-CD62L, and anti-TNF-α, these antibodies were obtained from eBioscience (San Diego, CA, USA). FITC-B540-labeled anti-CD3, APC-cy7-labeled anti-NK1.1, PE-YG582-labeled anti-CTLA4, PE-cy7-YG780-labeled anti-TIGIT, APC-R660-labeled anti-PD-1, V450-labeled anti-LAG-3, Percpcy5.5-B695-labeled anti-Tim-3, YG780-labeled anti-Granzyme B, and B540-labeled anti-perforin were obtained from Biolegend (California,USA) and BD (New York, USA). For the analysis of intracellular molecules, cells stained with anti-CD4 and anti-CD8 antibodies were fixed using Fix/Perm Buffer (eBioscience, San Diego, CA, USA) for 30 min, incubated with anti-mouse-IFN-γ and -TNF-α for 30 min at 4 °C, and then analyzed using flow cytometry.

Single-cell suspension of 1 × 10⁶ CT-26 and CD44⁺ CT-26 cells (namely, CCSCs) in 95 μl of PBS was incubated with Phycoerythrin (PE)-conjugated CD44 monoclonal antibody (eBioscience, San Diego, CA, USA) in the dark for 30 min at 4˚C and subsequently analyzed using Beckman Coulter FC500 Flow Cytometer with the CellQuest Pro software (BD Biosciences, San Jose, CA, USA) to determine the number of CD44⁺ cells. Similarly, PE-conjugated CD11c monoclonal antibody (eBioscience) was used to determine the number of CD11c⁺ cells in mononuclear cells isolated from bone marrow on day 0 and immature BMDCs from day 7 of culture.
The number of CD80, CD86, MHC-I, and MHC-II cells in unpulsed DCs and DCs pulsed with DRibbles (20 μg/ml) or lysate (20 μg/ml) were determined using anti-CD86, anti-CD80, anti-MHC-I, and anti-MHC-II antibody (all from eBioscience).
The percentages of CD8⁺ IFN-γ⁺ cells in total splenic lymphocytes of each experimental and control group were determined using anti-CD8 (eBioscience) and anti-IFN-γ (Cell signaling technology, Inc., Danvers, MA, USA) antibody.