Pe anti mouse cd4

Single cell suspensions from the spleen and liver were acquired according to the methods previously described (21 (link)) and analyzed using flow cytometry. Antibodies used for flow cytometry staining including Percp-Cy5.5-anti-mouse-CD45.1, APC-Cy7-anti-mouse-CD11b, Percp-Cy5.5-anti-mouse-NK1.1, BV650-anti-mouse-H2-Kb were purchased from BD Bioscience (SanDiego, CA, USA); purified anti-mouse-CD16/32, APC-anti-mouseCD43, PE-anti-mouse-NKp46, APC-anti-mouse-CD107, FITC-anti-mouse-NKG2D, PE-anti-mouse-IFN-γ, PE/Cy7-anti-mouse-TNF-α, FITC-anti-mouse-CD62E, PE-anti-mouse-CD4, PE/Cy7-anti-mouse-CD44, APC/Cy7-anti-mouse-CD62L, Pacific Blue-anti-mouse-CD8a, FITC-anti-mouse-CD69 were purchased from Biolegend (San Diego, CA, USA); PE-Cy7-anti-mouse-Granzyme B, APC-anti-mouse-Perforin were purchased from eBioscience (San Diego, CA, USA). Recombinant Mouse E-Selectin, P-Selectin, and L-Selectin chimera were purchased from Biolegend (San Diego, CA, USA) for detecting the binding abilities. Samples were detected on a NovoCyte Flow Cytometer (ACEA Biosciences, San Diego, CA, USA) and data were analyzed by using Flowjo software (Flowjo, Ashland, OR, USA).

Complete Freund’s adjuvant (CFA) is provided by Chondrex Company (Woodinville, USA). PE anti-mouse CD4, FITC anti-mouse CD8a, and PeCy7 anti-mouse OX40 antibody were supplied by BioLegend (San Diego, CA). InVivo MAb anti-mouse OX40 (clone: OX-86) and InVivoPlus rat IgG2a isotype control (clone: 2A3) were supplied by BioXCell (West Lebanon, USA). IRDye® 680RD NHS Ester Infrared Dye was supplied by Li-cor (Lincoln, USA). Fixable Viability Stain 780 was purchased from BD Pharmingen (San Diego, USA). Dimethyl sulfoxide (DMSO) Hybri-Max (TM) sterile-filtered, BioReagent was purchased from Sigma (St. Louis, MO, USA). AbC™ total antibody compensation bead kit was acquired from Thermo Fisher (Waltham, MA, USA). Paraformaldehyde, 4% and phosphate buffered solution (PBS) was supplied by Solarbio (Beijing, China). Fetal bovine serum (FBS) was purchased from Invitrogen (New York, America).

For surface staining, the cell suspension was incubated with fluorescently labeled antibody at room temperature for 20 minutes. For CD206 staining, the samples were fixed and permeabilised with BD Cytofix/Cytoperm Fixation/Permeabilization Solution kit (BD Biosciences, US), and then incubated with fluorescently labeled antibody at 4 °C for 35 minutes. For intracellular TNF-α staining, the samples were stimulated by LPS (100 ng/ml, Beyotime) for 4 h, fixed and permeabilised, and then incubated with fluorescently labeled antibody at 4 °C for 35 minutes. Bacteria were stained using the BacLight™ Red kit (Invitrogen, B-35001). miRNA was labelled with fluorescein amidites (FAM). The monoclonal antibodies used were as follows: APC-Cy7-anti-Mouse F4/80, Pacific Blue-anti-Mouse CD11b, FITC-anti-Mouse CD11c, APC-anti-Mouse CD206, APC-Cy7-anti-Mouse TCRβ, APC-anti-Mouse NK1.1, PerCP-Cy5-5-anti-Mouse CD45, PE-Cy7-anti-Mouse CD3, PE-anti-Mouse CD4, FITC-anti-Mouse CD8, APC-anti-Mouse TNF-α, all purchased from Biolegend; FITC-anti-Human CD86 (20 μl/ test), APC-anti-Human CD206 (20 μl/test), purchased from BD Pharmingen. Flow cytometry or Image Stream was conducted using a BD FACS CantoII or Millipore ISX with fluorochrome-conjugated cells, and the data was analysed using FlowJo software version 10.4 or IDEAS version 6.0.

For evaluating the frequency of Treg cells, splenocytes were first surface stained with PE anti-mouse CD4 (Biolegend) and FITC anti-mouse CD25 (Biolegend). After permeabilization with Foxp3 buffer set (BD Biosciences), cells were incubated with PerCP/Cy5.5 anti-mouse Foxp3 for intracellular staining. Finally, Data were acquired and analyzed with FACSCalibur flow and flowJo software (Version 10).

After the spleens were grinded into single cells and red blood cells were lysed, CD11c⁺ DCs were enriched with CD11c Microbeads UltraPure (Miltenyi Biotech, cat. 130-108-338, Cambridge, MA, USA). 2 × 10⁵ CD11c⁺ DCs were incubated with 600 µg Flag-tagged Xcl1-S, S or vector plasmid transfected 293T cell lysate for 30 min. Then, the cells were washed two times with PBS and subjected to FITC anti-Flag antibody (Abcam, cat. ab117505) for 30 min measured by flow cytometry using a FACS CANTO TWO flow cytometer (BD Biosciences). 1 × 10⁶ C57BL/6 mice splenocytes were plated on a 24-well plate supplied with overlapping peptides covering the SARS-CoV-2 S protein for 18 h at 37 °C in 5% CO₂ to stimulate T cell. Mice splenocytes were collected, and intracellular cytokine levels were measured by flow cytometry using a FACS CANTO TWO flow cytometer (BD Biosciences) after permeabilization and fixation of the cells (BD Biosciences). Cells were stained using the following antibodies from Biolegend: FITC anti-mouse CD8a, PE anti-mouse CD4 and APC anti-mouse IFN-γ. Finally, the results were examined with FlowJo software (FlowJo LLC, Ashland, OR, USA).

At the end of the experiment, excised MC38 tumors from mice treated with CBPA (10 mg/kg) or the vehicle control were digested into single-cell suspensions. Cells were washed, resuspended in FACS buffer, and used as antibodies against surface antigen staining for 30 min at 4 °C with FITC anti-mouse CD3 (Cat. 100203, BioLegend, San Diego, CA), PE anti-mouse CD4 (Cat. 100407, BioLegend, San Diego, CA), and PE anti-mouse CD8 (Cat. 100707, BioLegend, San Diego, CA). Cells were then fixed and permeabilized, stained for perforin (Cat. 154303, BioLegend, San Diego, CA) and granzyme B (Cat. 396413, BioLegend, San Diego, CA), and analyzed by flow cytometry.