Anti cd69 fitc

Cells were washed in flow cytometry staining buffer (BD Pharmingen), treated with Fc block (BD Bioscience) and stained for flow cytometric analyses of extracellular markers using fluorochrome‐conjugated antibodies according to the manufacturer's instructions. Antibodies included anti‐CD19‐PE or anti‐CD19‐FITC, anti‐CD5‐BV421 and the activation markers anti‐HLA‐DP, DQ, DR Alexa Fluor 647 or anti‐HLA‐DP/DQ/DR‐FITC, anti‐CD86‐PerCP‐CY5.5, anti‐CD69‐FITC and anti‐CD40‐PE‐Cy7 (BioLegend); markers of maturation anti‐CD38‐FITC or anti‐CD38‐APC and CD24‐APC‐Cy7 (BioLegend); or CD22‐FITC and sialic acid‐binding immunoglobulin‐like lectin (Siglec)‐10‐APC (BioLegend). Immunoglobulin isotype‐matched control antibodies were used to confirm specificity of staining. Extracellular staining was conducted for 30 min in the dark at 4°C, before the cells were washed and analysed by flow cytometry. To correct for inter‐assay variation, ratios were calculated of staining intensity in treated versus control cultures.

Jurkat-PD1 cells 2 × 10⁵/ml with or without wild-type Raji cells or Raji-PD-L1 cells (4 × 10⁵/ml) in 24-well plates were treated with YIV-906 with or without SEE (10 ng/ml and 30 ng/ml), for 48 h. Anti-CD69-FITC (BioLegend, Cat#310904) and anti-PD1-PE (BioLegend, Cat#A17188B) in PBS+1%BSA was used to stain the cells. anti-PD1-PE was used to separate Jurkat-PD1 cells from mixed cells. The expression of CD69 in the FITC channel was presented as median fluorescence or total Jurkat cell population percentage using a laser flow cytometer (BD, Accuri 6 plus). Cell death of Raji cells (co-cultured with Jurkat-PD1-CAR-CD-19 cells/Raji cells at a 2:1 ratio) was determined by gating CD19⁺ Raji cells (Anti-CD19-perCP, BioLegend, #30228) and Annexin V-PE (BioLegend, #640947) positive and/or Helix-NR positive cells. Each data point represents the average mean of four experiments of triplicate samples of flow cytometry assays.
Following the treatment, the medium was collected for IL2, IFNg, and IL10 detection using fluorescence bead array (BioLegend: LEGENDplex assays, Human CD8/NK Panel, Cat#740267) according to the to the manufacturer’s instructions.

₅₃₃IYSTVASSL₅₄₁ (IVL_533-541)-tetramer was bought from MBL (cat. NO.TS-M520-1), anti-CD8α depletion antibody (clone: YTS 169.4). Rat IgG2b isotype antibody (clone: LTF-2) was purchased from Bio X Cell. Antibodies for flow cytometry included anti-CD3-APC-eFluor780 (clone: 17A2; eBioscience), anti-CD4-Pacific Blue (clone: GK1.5; BioLegend), anti–CD8α-PerCP/Cy5.5 (clone: 53–6.7; BioLegend), anti-CD11a-PE/Cy7 (clone: 2D7; BD Bioscience), anti–CD44-Alexa Fluor 700 (clone: IM7; BioLegend), anti-CD45-APC-eFluor780 (clone:30-F11;Biolegend), anti-CD62L-eVolve 605 (clone: MEL-14; eBioscience), anti-CD127-PE & PE/Cy7 (clone: A7R34; BioLegend), anti-KLRG1-FITC & BV510 (clone: 2F1/KLRG1; BioLegend), anti-CD103-APC (clone: 2E7; BioLegend), anti-CD69-FITC (clone: H1.2F3; BioLegend) and anti-IFNγ-APC (clone: XMG1.2; BioLegend), anti-Eomes-PE & PE/Cy7 (clone: Dan11mag; eBioscience).

Tumor tissues, lymph nodes, and spleens were collected from mice after SDT and homogenized into single-cell suspensions following the established protocol (66 (link), 67 (link)). Then, the cell suspensions were stained with different antibodies according to the standard protocol. DCs in lymph nodes were stained with anti-CD11c–PE-Cy7 (BioLegend, catalog number 117318), anti-CD80–FITC (BioLegend, catalog number 104706), and anti-CD86–allophycocyanin (BioLegend, catalog number 105012) and analyzed by FCM (Thermo Fisher Scientific, Attune NxT).
To systematically investigate the in vivo antitumor immune responses against tumor metastasis, suspensions in tumors were stained with anti-CD3–PE (BioLegend, catalog number 100308), anti-CD8a–PerCP (BioLegend, catalog number 100732), and anti-CD4–allophycocyanin (BioLegend, catalog number 100412) antibodies. To further analyze T cells in spleens, cells were stained with anti-CD3–PE (BioLegend, catalog number 100308), anti-CD8a–PerCP (peridinin chlorophyll protein) (BioLegend, catalog number 100732), anti-CD4–allophycocyanin (BioLegend, catalog number 100412), anti-CD44–FITC (BioLegend, catalog number 103006), or anti-CD69–FITC (BioLegend, catalog number 104506). All these antibodies used in our experiments were in a 1:100 dilution from the stock solutions.

Peripheral blood mononuclear cells (PBMCs) from 75 patients were selected and divided in 4 groups according to sex and genotype at rs12959006 (approximately 19 patients per group). Due to the low frequency of the minor homozygote, only major homozygote and heterozygote patients were included. Treatment effect was accounted for by including patients with the same treatment in every group. Samples were cultured in RPMI supplemented with 10% FBS, 1% penicillin-streptomycin and 1% L-glutamine (Sigma-Aldrich, Bremen, Germany), without stimulus or treated with 5 μg/ml Concanavalin A (Con A, Sigma-Aldrich, Bremen, Germany). Afterward, they were kept at 37°C and 5% CO₂ for 24 h. Cells were stained with anti-CD69-FITC, anti-CD3-PE, and 7-AAD to exclude non-viable cells (Biolegend, San Diego, CA, United States). Samples were processed and analyzed in a Cytomics FC500 flow cytometer with Kaluza 2.0 software (Beckman Coulter, Brea, CA, United States) and percentage of CD69⁺ cells and median fluorescence intensity (MFI) of CD69⁺ cells in CD3⁺ and CD3^– populations were measured. Final values were calculated by extracting the basal CD69 measures (without stimulus) to the samples treated with Con A.