Fitc anti mouse cd3

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.

Rat MOG35–55 peptides were purchased from Biosynth International (Naperville, IL, USA) and the purity of the peptide was >95%. The sequence of MOG35–55 was MEVGWYRSPFSRVVHLYRNGK. FITC anti-mouse CD3, PE/Cy7 anti-mouse CD4, APC/Cy7 anti-mouse CD44, PerCP/Cy5.5 anti-mouse CD11b antibodies, and LEAF Purified anti-mouse IL-6 were purchased from BioLegend (San Diego, CA,USA). Alexa Fluor700 anti-mouse CD45.2, PE anti-mouse CD69, APC anti-mouse CD25, PE anti-mouse IL-17A, and APC anti-mouse IFN-γ antibodies were purchased from eBioscience (San Diego, CA, USA). PE/Cy5 anti-mouse CD8α antibodies were purchased from BD Pharmingen (Basel, Switzerland). Goat anti-mouse ionized calcium-binding adaptor molecule-1 (IBA1) antibody was obtained from abcam (Cambridge, UK). Rat anti-mouse CD45, Rabbit anti-mouse CCL-2 antibody, and CCL-2 (rat recombinant) were purchased from AbD Serotec (Raleigh, NC, USA). Rabbit anti-mouse CCR2 antibody was purchased from Abcam (Cambridge, UK). 2-((1-benzyl-indazol-3-yl) methoxy)-2-methyl propionic acid (Bindarit) was synthesized by and obtained from Angelini (Angelini Research Center-ACRAF, Italy).

For detecting only BCSCs in cancers, cancer cells were stained with specific marker proteins using antibodies such as FITC anti-CD44 and APC anti-CD24 (BD, San Jose, CA, USA) to define CD44+/CD24-BCSCs. Single-cell suspensions were prepared from mouse tumor tissues or TDLNs to analyze the T-cells. The processes of tissue sample preparation are described in “Subcutaneous tumor and TDLN resection and sample preparation” section. Cells were stained with specific antibodies as APC anti-mouse CD8a, APC anti-mouse CD4, and FITC anti-mouse CD3 (BioLegend, San Diego, CA, USA) to define CD8+/CD3 + cytotoxic T-cells or CD4+/CD3 + helper T-cells. The samples were analyzed by flow cytometry (Accuri C6, BD Biosciences, East Rutherford, NJ, USA).

To measure PD­L1 levels on the cell surface of tumour cells treated with vehicle, cisplatin, radiation, JQ1, IFN‐γ (20 ng/ml,²⁴ PeproTech), or the combinations for the different time periods were trypsinized and harvested for staining with streptavidin‐phycoerythrin (PE)‐conjugated anti‐human PD‐L1(329706, Biolegend). Tumour tissues were collected, cut into small pieces, digested with a mixture of collagenase, DNase and hyaluronidase for preparing single‐cell suspension and then treated with Cell Stimulation Cocktail (00‐4975‐03, eBioscience) as instructed. Cells were washed, re‐suspended in fluorescence‐activated cell sorting buffer at 4°C and then stained with fluorescent‐conjugated antibodies and appropriate isotype controls for multicolor flow cytometry. For cell surface staining, the following antibodies were used: fluorescein isothiocyanate (FITC) anti‐mouse CD3 (100204, Biolegend), PE/Cy7 anti‐mouse cluster of differentiation 4 (CD4) (100422, Biolegend), APC anti‐mouse CD8 (100712, Biolegend), Brilliant Violet 605 anti‐mouse NK1.1 (108739, Biolegend) and PE anti‐mouse PD‐L1(124308, Biolegend). The collected live single cells were fixed and treated with permeabilization buffer before intracellular staining with Brilliant Violet 421 anti‐mouse IFN‐γ (505830, Biolegend).

CD3⁺CD4⁺ T cells, CD3⁺CD8⁺ T cells and CD3⁺CD4⁺FOXP3⁺ T cells, CD11c ⁺CD86⁺CD80⁺ dendritic cells as well as CD45⁺CD11b⁺F4/80⁺CD206⁺ M2 like macrophage and CD45⁺CD11b⁺F4/80⁺CD86⁺ M1 like macrophage in the tumor tissues or spleen were isolated and analyzed using flow cytometry. The antibodies involved in the experiment include FITC anti-mouse CD11c Antibody (Biolegend, Cat# 117306, Clone No. N418), PE anti-mouse CD86 Antibody (Biolegend, Cat# 105007, Clone No.GL-1), APC anti-mouse CD80 Antibody (Biolegend, Cat# 104714, Clone No.16-10A1), FITC anti-mouse CD3 (Biolegend, Cat# 100203, Clone No.17A2), PE anti-mouse CD4 (Biolegend, Cat# 100407, Clone No.GK1.5), APCanti-mouseCD8a (Biolegend, Cat# 100712, Clone No.53-6.7), AlexaFluor488 anti-mouse FOXP3 (Biolegend, Cat# 320012, Clone No.150D), FITC anti-mouse/human CD11b Antibody (Biolegend, Cat# 101205, Clone No. M1/70), APC anti-mouse CD45 (Biolegend, Cat# 103112, Clone No.30-F11), PerCP anti-mouse F4/80 Antibody (Biolegend, Cat# 123126, Clone No.BM8) and PE/Cyanine7 anti-mouse CD206 (Bioegend, Cat# 141720, Clone No.C068C2). The antibody concentration was 1:100 diluted with PBS.
IL-2, IL-12p70, TNF-α, and IFN-γ in primary tumors were also examined with ELISA kits. And tumors were stained for immunofluorescence of CD3⁺CD4⁺ and CD3⁺CD8⁺ proliferated CTLs in 4T1 tumor tissue slices of the primary tumor.

The radioisotope ²¹¹At was produced via ²⁰⁹Bi (α, 2n) ²¹¹At reaction through CS-30 cyclotron according to the published protocol [25 (link)]. Bovine serum albumin (BSA), Manganese chloride tetrahydrate (MnCl₂·4H₂O), and sodium hydroxide (NaOH) were from Sigma-Aldrich. Roswell park memorial institute (RPMI) 1640 medium, penicillin-streptomycin, and fetal bovine serum (FBS) were obtained from Gbico. The cell counting kit-8 (CCK-8) was obtained from Biosharp. The anti-mouse PD-L1 antibody was obtained from BioXcell (clone:10F.9G2). APC anti-mouse CD45(Catalog: 103111), FITC anti-mouse CD3 (Catalog: 100203), BV421™ anti mouse CD4(Catalog: 100437), APC/Cy7 anti-mouse CD8a (Catalog: 100714), PE anti-mouse FOXP3 (Catalog: 126403), FITC anti-mouse CD11c (Catalog: 117305), PE anti-mouse CD86 (Catalog:105007), APC anti-mouse CD80 (Catalog: 104713), PE/Cy7 anti-mouse CD45 (Catalog: 103113), PE anti-mouse CD44(Catalog:103023) and BV421- anti-mouse CD62L (Catalog: 104435) antibodies were obtained from Biolegend, Tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) ELISA kit was purchased from Multisciences Biotech, Co., Ltd.