Anti cd8a pe

Intracellular flow cytometry analysis was performed as previously described (22 (link)). Briefly, 5 × 10⁶ splenocytes were plated in each well of a 24-well plate and incubated with either CMFO (10 µg/mL) or PPD (10 µg/mL) in the presence of 1 µg/mL anti-CD28/CD49d (eBioscience CA, USA). RPMI1640 medium and cell stimulation cocktail (eBioscience, CA, USA) were used as negative and monitoring controls, respectively. Cells were stained with surface markers, including anti-CD4 PE Cy7, anti-CD8a PE, anti-CD44 APC-eFluor^® 780, anti-CD62L FITC mAbs, and intracellular markers anti-IFN-γPerCP-Cy5.5 and anti-IL-2 APC mAbs (all from eBioscience, CA, USA). The stained cells were analyzed by an LSRII multicolor flow cytometer (BD Biosciences, CA, USA). The absolute number of CMFO-specific CD4⁺ or CD8⁺ IFN-γ positive T_EM (effector memory T cells, CD62L^loCD44^hi) and CD4⁺ or CD8⁺ IL-2 positive T_CM (central memory T cells, CD62L^hiCD44^hi) cells were analyzed with FlowJo software (Tree Star Inc., OH, USA). The results are represented as mean ± SD per group (n = 6).

The number of MDSCs, NKT cells, and T cells subsets were detected using FCM. Tumor tissues were cut into small fragments, digested with 0.25% Trypsin for 30 min, and then filtered using 70 μm cell strainers. This single-cell suspension was then centrifuged at 1500 rpm for 5 min. The supernatant was discarded and the cell concentration was adjusted to 5 ×10⁸/ml. The cell suspension was then washed using a mouse tumor-infiltrating lymphocyte separation medium (CW0049S; CWBIO, Jiangsu, China) and centrifuged at 1500 rpm for 15 min to obtain the lymphocytes. Lymphocytes with a concentration of 2 × 10⁶/ml were collected and washed with PBS, followed by staining with anti-CD11b FTIC (No. 557397; BD, USA), anti-LY6G (No. 553989; BD, USA) and LY6C PE (No. 553126; BD, USA) (Gr-1), anti-CD3e FTIC (No. 46003 280; eBioscience, USA), anti-CD49a PE (No. 130107632; BD, USA), anti-CD4 FTIC (No. 11004282; eBioscience, USA), anti-CD8a PE (No. 11008182; eBioscience, USA), and anti-CD3 PerCP-CY 5.5 (No. 46 003280; eBioscience, USA), along with the appropriate isotype controls. The cells were analyzed using a flow cytometry FACSCalibur (BD, USA).

Small intestine and colon tissues were incubated at 37 °C in PBS containing 2% FBS and 5 mm EDTA for 25 min. The remaining tissue was cut into small pieces and digested in PBS containing 2% FBS, Collagenase IV (0.5 mg mL⁻¹; Thermo), and DNase I (10 U mL⁻¹; Sigma‐Aldrich) and then incubated at 37 °C for 45 min. Single cell suspensions were stained with anti‐CD45‐Alexa Flour 700 (eBioscience, 30‐F11, 56‐0451‐82, 1:400), anti‐CD4‐APC‐Cy7 (Biolegend, GK1.5, 100 414, 1:400), anti‐CD8a‐ PE (eBioscience, 53–6.7, 12‐0081‐83, 1:400), anti‐NK1.1‐PE‐Cy7 (eBioscience, PK136, 25‐5941‐82, 1:400), anti‐CD19‐APC (eBioscience, 1D3, 17‐0193‐80, 1:400), anti‐CD11b‐FITC (Biolegend, 101 206, M1/70, 1:400), anti‐CD11c‐PE (eBioscience, N418, 12‐0114‐82, 1:400), and FVD eFlour 506 (eBioscience,1:1000) for FACS analysis (Thermo). All flow cytometry analyses were performed on an Attune NxT Flow Cytometer (Thermo Fisher Scientific), and data were analyzed using FlowJo 10 software.

The maturation of DCs was examined by staining with fluorescence‐conjugated antibodies including anti‐CD11c‐FITC (eBioscience, catalog: 11‐0114‐85), anti‐CD86‐APC (eBioscience, catalog: 17‐0862‐82), and anti‐MHC Class II‐PE (eBioscience, catalog: 12‐5321‐82). To analyze the T‐cell subsets in spleens and draining lymph nodes of the immunized mice, single cell suspensions prepared from these samples were examined by flow cytometry. The following primary antibodies were used: anti‐CD3e‐PerCP‐Cyanine5.5 (eBioscience, catalog: 45‐0031‐82), anti‐CD8a‐PE (eBioscience, catalog: 12‐0081‐82), anti‐CD4‐FITC (eBioscience, catalog: 11‐0041‐85), anti‐CD25‐APC (eBioscience, catalog: 17‐0251‐82), anti‐Foxp3‐PE (eBioscience, catalog: 12‐4771‐82), anti‐IFN‐γ‐FITC (BD Bioscience, catalog: 554 411), anti‐TNF‐α‐Alexa Fluor 647 (BD Bioscience, catalog: 557 730), and anti‐CD16/CD32 (eBioscience, catalog: MA5‐18012). Flow data were acquired on a CytoFLEX and analyzed using CytExpert (Beckman Coulter, USA) and FlowJo (TreeStar, USA) softwares.