Mhc 1

Cells were detached from culture dish using HyQtase and blocked with Fc (CD32/16) at RT for 15 min, followed by incubating with primary antibodies conjugated with fluorescence at 4 °C for 30 min in darkness [37 (link)]. The cells were then assessed using BD FACS Canto II, and the data was analyzed using FlowJo software (TreeStar). Analyses included CD117 (Abcam, Cat. ab5506, 1:10), Sca1 (BioLegend, Cat. 108111, 1:50), CD73 (BioLegend, Cat. 127209, 1:50), CD90.2 (BioLegend, Cat. 105307, 1:50), CD105 (BioLegend, Cat. 120407, 1:50), CD34 (BioLegend, Cat. 128611, 1:50), CD31 (BioLegend, Cat. 102407, 1:50), CD45 (BioLegend, Cat. 103111, 1:50), CD11b (BioLegend, Cat. 101207, 1:50), MHC I (eBioscience, Cat. 17-5957-80, 1:50), and MHC II (eBioscience, Cat. 17-5957-82, 1:50).

The expression of mesenchymal stem cell surface antigens (CD105, CD73, CD90 and CD34)(antibodies all from abcam, Cambridge, UK) and co-stimulatory molecules (MHC-I, MHC-II, CD80, CD86,andCD40) (antibodies all from eBioscience, UK) were analyzed by flow cytometry. Briefly, appropriate 0.5×10⁶ cells were washed and resuspended in 100μl of the buffer(PBS containing 2% FBS). Saturating concentrations (1:50) of fluorescein isothiocyanate (FITC)-conjugatedanti-CD105, anti-CD73,anti-CD90,anti-CD34, anti-CD80, anti-MHC-I, anti-MHC-II, or phycoerythrin (PE)-conjugated anti-CD40, anti-CD86were added into each of tubes and incubated on ice for 30 minutes. After two washes with PBS, all cell suspensions were resuspended in PBS containing 2% FBS for analysis on a flow cytometer (FACSCalibur; BD Biosciences) using the CellQuest software.

To determine mature molecular marker expression on the surface after infection with NDV, cells were harvested at 48 hpi and stained with FITC or APC-labeled CD11c, PE-labeled CD80, MHC-I, MHC-II, CCR5, or CCR7, PE or PE-Cy5-labeled CD86 and PE or FITC-labeled CD40 antibodies (eBioscience, San Diego, CA, USA). After staining, maturity of infected DCs was determined by proportions of cells expressing markers (%) using flow cytometry.
Detection of apoptosis was performed using the Annexin V Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. Following infection with NDV, DCs or T cells were collected and incubated with CD11c-APC or CD3-PE-Cy5 antibodies, and then labeled with Alexa Fluor^® 488 annexin V and PI working solution. Thereafter, apoptosis of cells induced by NDV was analyzed by flow cytometry.
For intracellular cytokine staining, lymphocytes or DCs were pretreated with 2 mM Brefeldin A (to inhibit intracellular protein transport) for 4 h. After harvesting, cells were stained with CD11c-APC or CD3-PE-Cy5 and CD4-FITC/CD8-FITC antibodies. Next, cells underwent intracellular fixation and permeabilization and were subsequently stained with IL-10-PE, IFN-γ-PE, or IL-4-PE antibodies.

Flow cytometry was performed on a FACS LSRFortessa flow cytometer with Diva software (BD Bioscience, USA). For DC analysis in vitro and in vivo, the cells were stained with CD11b (clone: M1/70, BioLegend), CD11c (clone: HL3, BD), CD80 (clone: 16-10A1, eBioscience), CD86 (clone: GL-1, BioLegend), MHCI (clone: AF6.88.5.5.3, eBioscience), MHCII (clone: M5/114.15.2, eBioscience), PDCA-1 (clone: eBio927, eBioscience). For monocytes analysis in vivo, the cells were stained with CD11b (clone: M1/70, BioLegend), Ly6G (clone: RB6-8C5, eBioscience), Ly6C (clone: HK1.4, BioLegend), CCR2 (clone: SA203G11, BioLegend). For intracellular cytokine detection, the splenocytes were stimulated with rHBsAg (10 μg/mL) for 18 h. All samples included addition of 10 μg/ml Brefeldin A (BFA, BD) for the last 6 h of incubation. Tests included: PMA/ionomycin (100 ng/mL/1 μg/mL) as positive control; Th1 (CD4 (clone: RM4-5, BioLegend); IFN-γ (clone: XMG1.2, BioLegend); Th2 (CD4 (clone: RM4-5, BioLegend); anti-IL4 (clone: 11B11, BioLegend)); Tc1 (CD8 (clone: 53-6.7, eBioscience); anti-IFN-γ (clone: XMG1.2, BioLegend)). Isotype-matched control mAb were used to identify non-specific background staining.

Following 8–14 weeks post orthotopic transplants, or ~8 weeks post i.c. or i.v. injections, primary tumors and peripheral tissues including lungs, lymph nodes, bone marrow, peripheral blood, and brains were harvested, dissociated to single cells and stained with fluorescently conjugated antibodies for CD298 (Biolegend, Cat. no. 341704) and MHC-I (eBioscience, Cat. no. 17-5957-82), and flow cytometry was used to analyze and quantify disseminated CD298⁺MHC-I⁻ PDX cells using the BD FACSAria Fusion cell sorter (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) as described previously^{26 (link)}. Primary tumor volumes were calculated using caliper measurements with the equation: volume of an ellipsoid = 1/2(length × width²). Whole-mount images of primary tumors and lungs were taken with a Leica MZ10 F modular stereomicroscope (Leica Microsystems, Buffalo Grove, IL, USA).

The C57BL/6 mice at the age of 18–22 weeks were killed and removed the epididymal adipose tissues and minced it into small pieces (∼2 mm). Then the pieces of adipose tissues were collected and incubated them at 37°C for about 2 hours in collagenase II with gentle stirring. The digested tissues were centrifuged at 500 g for 5 min. The resultant pellet containing the total lymphocytes were re-suspended in PBS and filtered it through a 100 M mesh, and we finally re-suspended them in 100 μl PBS supplemented with 5% FBS. All these cells were incubated with FITC-CD11c, PE-CD40, CD80, CD86, MHCI, MHCII (eBioscience, San Diego, CA) conjugated antibodies or respective isotype controls for 30 min at 4°C. All the staining was performed according to manufacturers' protocol. Flow cytometry was performed using FACS Calibur instrument (Becton Dickinson) and WinMDI 2.8 software was used to analyze the data.