Anti cd3 percp cy5

Gastrointestinal biopsies were immediately transferred to a dithiothreitol/EDTA solution (Sigma, Hamburg, Germany, and Life Technologies, Darmstadt, Germany) and incubated for 15 min at 37 °C. The samples were finely sliced and digested in a collagenase A solution (Roche, Mannheim, Germany) for 1 h at 37 °C. The resulting cell suspension was passed through a 70-µm cell strainer (BD Biosciences, San Jose, CA, USA) and collected in a cRPMI-10 filled tube (Life Technologies, Darmstadt, Germany). After centrifugation, the pellet was resuspended in PBS (Life Technologies, Darmstadt, Germany) and cell viability was confirmed by staining an aliquot with 0.4 % trypan blue (Sigma, Hamburg, Germany) for microscopy.
For the quantification of CD3⁺CD4⁻CD8⁺ T cells (CD8⁺ T cells) in lamina propria by flow cytometry, the following monoclonal antibodies were used: anti-CD3 PerCP-Cy5.5 (eBiosciences, USA) and anti-CD4 PE and anti-CD8 V500 (BD Biosciences, San Diego, CA, USA). Positive signals were defined by the use of isotype controls and the fluorochrome minus one (FMO) method.

Anti-ULBP4 mAb (clone 709116) was purchased from R&D (Minneapolis, MN, USA). The ULBP4-specific mAb DUMO1 was previously described [32 (link)]. Anti-CD3-PerCP-Cy5.5 and fixable viability dye(FVD)-eFluor506 were from eBioscience (SanDiego, CA, USA), anti-CD19-APC-Cy7, anti-CD14-FITC, anti-NKp46-PE-Cy7, streptavidin-BV421 (SA-BV421) and goat-anti-mouse-IgG-PE (GaM-PE) were from Biolegend (SanDiego, CA, USA).

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).

Mouse cells were preincubated with purified antimouse CD16/CD32, and human cells were incubated with FcR-blocking reagent (BD Biosciences). Isolated cells were stained with labeled antibodies in phosphate-buffered saline (PBS) with 1% bovine serum albumin (BSA). Dead cells were excluded based on staining with Live/Dead fixable dye (eBioscience). Forkhead box P3 (FOXP3) fixative solution (eBioscience) was used for FOXP3 staining. Prepared samples were analyzed using a flow cytometer (FACSCalibur or FACSAria; BD Biosciences). Data were analyzed using FlowJo software (TreeStar, Ashland, OR, USA). The following beads and antibodies were used: OneComp eBeads (eBioscience), antimouse CD11c Brilliant Violet 421 (BioLegend), antimouse CD11b PE-cy7 (eBioscience), GR1-APC (eBioscience), LY6C-APC-eFluor (eBioscience), F4/80 PE (eBioscience), NK-1.1 Percp-Cy5.5 (eBioscience), anti-CD45 FITC (eBioscience), Live/Dead Aqua (eBioscience), anti-CD4 eFluor 450 (eBioscience), anti-CD8 APC eFluor 780 (eBioscience), anti-CD3 Percp-Cy5.5 (eBioscience), anti-CD19 PE-CY7 (eBioscience), anti-CD25 APC (eBioscience), anti-Foxp3 PE (eBioscience), and anti-CD3 APC (BioLegend).