Apc anti mouse cd3

Cell suspensions were counted and incubated in 1% BSA/DPBS for 30 min at 4 °C to block non-specific binding. The cells were incubated with FITC anti-mouse CD45 (157,214, Biolegend), APC-anti-mouse CD3 (100,236, Biolegend), PE/CY5.5 anti-mouse CD4 (100,434, Biolegend), and PE anti-mouse CD8a (100,708, Biolegend) and subsequently permeabilized with a suitable buffer (Biyuntian, China) ; moreover, they were incubated overnight with PE anti-mouse Foxp3 (126,404, Biolegend) and BV421 anti-mouse T-bet (644,815, Biolegend) antibodies at 4 °C for intracellular staining. To analyze cytokine secretion, the cells were stimulated with Monensin (420,701, Biolegend) and a cell activation cocktail (423,301, Biolegend), followed by surface staining, fixation, and permeabilization as described. The cells were the incubated overnight with PE anti-mouse INF-γ (505,808, Biolegend), BV421 anti-mouse IL-4 (504,119, Biolegend), and PE/CY7 anti-mouse IL-17 A (506,922, Biolegend) antibodies at 4 °C. The data was analyzed using Flowjo software (Tree Star Inc., San Carlos, CA).

Antibodies used included rabbit anti-human PD-L1 (#13684, Cell Signaling Technology, Beverly, MA, USA; 1:1000 for Western blot), anti-Cleaved Caspase-3 (#9664, Cell Signaling Technology; 1:250 for IHC), Rabbit anti-human AhR (#83200, Cell Signaling Technology; 1:50 for ChIP), goat anti-mouse PD-L1 (#AF1019, R&D, Minneapolis, MN, USA; 10 µg/mL for IHC), anti-Ki67 (#ab15580, Abcam, Cambridge, MA, USA; 1:500 for IHC), TTF1 (#ab76013, Abcam, Cambridge, MA, USA; 1:200 for IHC), anti-β-Actin (#A1978, Sigma, St. Louis, MO, USA; 1:5000 for WB), APC anti-mouse CD3 (#100235, Biolegend, San Diego, CA, USA; 1:20 for flow cytometry), PE/Cy7 anti-mouse CD8a (#100721, Biolegend; 1:20 for flow cytometry), APC/Cy7 anti-mouse CD4 (#100413, Biolegend; 1:20 for flow cytometry), PE anti-mouse CD45 (#103105, Biolegend; 1:20 for flow cytometry), PE/Cy7 anti-mouse PD-L1 (#124313, Biolegend; 1:20 for flow cytometry), PE anti-mouse PD-1 (#135206, Biolegend; 1:20 for flow cytometry), FITC anti-mouse B220 (#103205, Biolegend, San Diego, CA, USA; 1:20 for flow cytometry), In Vivo Plus anti-mouse PD-L1 (#BP0101), In Vivo Plus Rat IgG 2b Isotype Control (Clone: LTF-2, #BP0090) were purchased from BioXcell (West Lebanon, NH, USA). Benzo(a)pyrene (#B1760) and Alpha-Naphthoflavone (ANF; # N5757) were purchased from Sigma.

Splenocytes from infected C57BL/6 (3 months post-infection) mice were plated into 24-well plates at a cell density of 10⁵ cells/mL in RPMI medium supplemented with 10% FBS containing 2 μg/mL of Con A in the absence or presence of mDC or tDCs (1:10 ratio) for 72 h. Quantitative evaluation of the exponential cell expansion was estimated by the carboxyfluorescein succinimidyl ester–CFSE assay (Invitrogen/Molecular Probes). CFSE staining was performed according to methodology previously described (27 (link)). Before acquisition, cells were centrifuged and the pellet was washed twice with cold PBS and labeled with APC anti-mouse CD3 (Biolegend, San Diego, CA) diluted 1:100 for 15 min. Acquisition was performed using a BD LSRFortessa SORP cytometer and data were analyzed using FlowJo software (Tree Star, Ashland, OR). A total of 100,000 events were acquired.

Recombinant human TNFα was purchased from Sigma (St. Louis, MO, USA, 0609AFC25). The antibodies used were mouse anti-human Pacer (Novus, B01P, NBH00080183-B01P), anti-rabbit GAPDH (Cell Signaling Technology, Danvers, MA, USA, 2118s), anti-mouse PTGS2 (Cox2) (Beckton Dickinson, Franklin Lakes, NJ, USA, 610203) and custom anti-mouse Pacer manufactured by Abmart raised against a 14 aa peptide of the N-terminal domain of mouse Pacer [33 (link)]. Concanavalin A (ConA) (Sigma, SLBR2953) was purchased from Sigma (St. Louis, MO, USA, SLBR2953). SYTOX™ Green dyekit was purchased from Life Technologies (Darmstadt, Germany, S7020), and the antibody used for flow cytometry was APC anti-mouse CD3 (BioLegend, San Diego, CA, USA, 100236). Colitis-grade dextran sulfate sodium (DSS) (MP Biomedicals, Santa Ana, CA, USA, 160110) and C57BL/6 mice were acquired from the Jackson Laboratory and maintained under standard conditions in the animal facility of Universidad Mayor Faculty of Science. All animal procedures were approved by the Animal Welfare and Ethics Committee of Universidad Mayor (Protocol Number, 06/2016-13-2017(E1)).

After 4 days of SINV injection into the U-87MG subcutaneous tumor model, tumor tissue was taken in a 100 mm petri dish, and each sample was treated separately as follows: rinsed with 1× HBSS and then transferred to a 15 milliliter (mL) centrifuge tube after the tissue had been cut up with scissors. A total of 5 mL of 10× triple enzyme mix (1 g Collagenase IV, 100 mg Hyaluronidase, and 20,000 units DNase in 100 mL HBSS solution, filtered through a 0.22 micrometer filter, dispensed, and stored at −20 °C; all enzymes used were obtained from Yeasen) was added to the centrifuge tube and digested overnight at room temperature. The digestion was then suspended by adding RPMI 1640 medium containing serum (Thermo Fisher) and centrifuged at 300× g for 5 min. The precipitate was resuspended in 100 µL PBS to make a cell suspension, to which 2 µL of primary antibody was added and incubated on ice in the dark for 20 min. Cells were resuspended by adding 200 µL PBS, and 1 µL Zombie Aqua™ Fixable Viability Kit (Cat# 423101, Biolegend, San Diego, CA, USA) was added to exclude dead cells, incubated on ice for 3–5 min, and then analyzed by flow cytometry. The antibodies used in the analysis were as follows: APC anti-mouse CD3 (Cat#100236, Biolegend); PE anti-mouse CD49b (Cat#103506, Biolegend). The results obtained were analyzed using Flowjo, version 10.8.1 software.