Fitc anti mouse cd3 antibody

The perfused mouse brains were rinsed in Hank’s buffer solution to remove the surface foreign matter, and the brain tissues were cut to the size of rice grains with surgical scissors and added with 1 mg mL⁻¹ collagenase IV (Sigma-Aldrich) and 50 U mL⁻¹ DNase I (Sigma-Aldrich, 15 kU) in 1640 medium in 5 mL of tissue digest, placed on a 37 °C shaker, and fully digested for 1 h. The entire digested tissue homogenate was poured into a 70 µm nylon filter, and the tissue residue was filtered by grinding the tissue with the flat end of a syringe, the above cell suspension centrifuged (200 × g, 5 min) to remove the supernatant. The cells were blown well by adding staining buffer. CD3⁺CD8⁺ T cells were stained with FITC-anti-mouse CD3 antibody (dilution of 1:50, catalog number: 100203, clone: 17A2, Lot: N418 Biolegend) and APC anti-mouse CD8 antibody (dilution of 1:80, catalog number: 100711, clone: 53-6.7, Lot: B329662, Biolegend). Flow cytometry data was obtained from flow cytometry (BD Accuri C6 Plus flow cytometer) and analyzed with CytExpert (2.3) software and FlowJo (Version 10).

Mouse blood cells were harvested and divided into three parts. First, spleen cells were treated with 100 μL of 20 μg/mL FITC anti-mouse CD3 antibody (100306, BioLegend, San Diego, USA), 100 μL of 12.5 μg/mL Percp anti-mouse CD4 antibody (100407, BioLegend), and 100 μL of 12.5 μg/mL APC anti-mouse CD8 antibody (100732, BioLegend) at 25°C for 30 min, washed, then incubated with 100 μL of 10 μg/mL FITC goat anti-mouse IgG antibody (405305, BioLegend) at 25°C for 30 min in the dark. Second, blood cells were treated with 100 μL of 12.5 μg/mL Percp anti-mouse CD4 antibody (BioLegend), 100 μL of 25 μg/mL APC anti-mouse CD25 antibody (101910, BioLegend), and 100 μL of 12.5 μg/mL PE anti-mouse Foxp3 antibody (320008, BioLegend) via the same protocol described above. Third, blood cells were treated with 100 μL of 20 μg/mL FITC anti-mouse CD19 antibody (115506, BioLegend) using the same protocol described above. After incubation, cells were washed and resuspended in 0.5 mL of PBS/2% paraformaldehyde and quantified by flow cytometry (BD Calibur^TM).

CSPCM was provided by HeBei TaiFeng Biotechnology Co., Ltd. (Handan, China). CTX CAS#6055-19-2(C₇H₁₅Cl₂N₂O₂P·H₂O) was purchased from Source Leaf Biotechnology Co., LTD(Shanghai, China). Eastep® Super Total RNA Extraction kit was purchased from Promega (Beijing) Biotechnology Co., LTD (Beijing, China). PrimeScript™ RT reagent Kit with gDNA Easer, TB Green® Premix Ex Taq™ II(Tli RNaseH Plus) were purchased from Takara Biomedical Technology (Beijing) Co., LTD (Beijing, China). BCA Protein Assay Kit was purchased from Beijing ComWin Biotechnology Co., LTD. (Beijing, China). SDS-PAGE Gel preparation kit was purchased from Biomed Genentech Inc. (Beijing, China). Rabbit Anti-Foxp3 Polyclonal Antibody, Rabbit Anti-RORγt Polyclonal Antibody and Mice Anti-GAPDH Polyclonal Antibody were purchased from Beijing Bioss Biotechnology Co. LTD (Beijing, China). FITC anti-mouse CD3 Antibody, APC anti-mouse CD4 Antibody, PE anti-mouse IL-17A Antibody, FITC anti-mouse CD4 Antibody, APC anti-mouse CD25 Antibody, PE anti-mouse FOXP3 Antibody, True-Nuclear™ Transcription Factor Buffer Set, True Nuclear TM 4X Fix Concentrate, True Nuclear TM 10X Per and True Nuclear TM Fix Diluent were purchased from BioLegend, Inc (California, United States).

pNKs cocultured with DSCs or decidual HESCs were collected, and the expression of CD45, CD3, CD56 and CD16 was analyzed by flow cytometry assays. Briefly, the collected pNKs were washed twice with PBS and stained with PerCP anti-human CD45 antibody (Biolegend, San Diego, CA, USA), FITC anti-human CD3 antibody (Biolegend), BV421 anti-human CD56 antibody (Biolegend) and APC-Cy7 anti-human CD16 antibody (Biolegend) diluted in PBS with 2% FBS at 4 °C for 30 min. pNKs were then washed twice and analyzed by flow cytometry assays.
DICs isolated from decidual tissue of mice treated with DMSO or LDN57444 were collected, and the expression of CD45, CD3 and NK1.1 was analyzed by flow cytometry assays. The procedure was described above, and the antibodies used were APC-Cy7 anti-mouse CD45 antibody (Biolegend), FITC anti-mouse CD3 antibody (Biolegend) and BV421 anti-mouse NK1.1 antibody (Biolegend).

The Immunofluorescence (IF) staining procedure on tissue slides from paraffin blocks follows the protocol described previously,^[25] The primary antibodies used include anti‐EpCAM (Abcam, ab32392), anti‐Cytokeratin 19 (CK19) (Abcam, clone EP1580Y, ab52625), FITC‐conjugated anti‐α‐SMA (GeneTex, GTX72531), Spark YG 570‐conjugated anti‐mouse CD31 (BioLegend, 102531), FITC anti‐mouse CD3 Antibody (BioLegend,100204), Spark YG 570 anti‐mouse F4/80 (123159), PE‐conjugated anti‐human CD31 (BioLegend, 303106). Samples stained with anti‐EpCAM and anti‐CK19 primary antibodies were labeled with secondary antibody Alexa Fluor 647‐conjugated anti‐rabbit IgG (BioLegend, 406414). Images were taken by a Leica DM4B fluorescent microscope or a confocal microscope.

MC38 tumour-bearing mice were randomly divided into four groups and treated with PBS, α-PD1, PGN_4.9 nanoadjuvant, and α-PD1 + PGN_4.9 nanoadjuvant. PGN_4.9 nanoadjuvant was intravenously injected into the C57BL/6 mice (equivalent to 2 mg kg⁻¹ IMDQ) on days 3, 9 and 15. Simultaneously, mouse α-PD1 (100 μg/mouse, BioXcell) was administered intraperitoneally on days 0, 3, 6 and 9 (n = 11 mice for the PBS group; n = 10 mice for other groups). The tumour volumes were measured by electronic calipers every 2 days, and the survival curves were recorded according to Kaplan–Meier analysis. Besides, the tumour samples were stained with PerCP/Cy5.5 anti-mouse CD45 (103132, Biolegend, clone number: 30-F11, Dilution 1:100), FITC anti-mouse CD3 antibody (100204, Biolegend, clone number: 17A2, Dilution 1:50), PE anti-mouse CD4 antibody (100512, Biolegend, clone number: RM4-5, Dilution 1:80), APC anti-mouse CD8α antibody (100712, Biolegend, clone number: 53-6.7, Dilution 1:80), and BV421 anti-mouse CD25 (102043, Biolegend, clone number: PC61, Dilution 1:200) according to the manufacturer’s instructions. The proportions of CD3⁺CD8⁺ cytotoxic T lymphocytes and CD4⁺CD25⁺ regulatory T lymphocytes were obtained by flow cytometry.