Anti cd3 antibody

Brains were dissected and post‐fixed in 4% PFA overnight. Samples were then transferred to a 30% sucrose solution until fully equilibrated. Midbrain tissues were coronally sectioned (35 mm). For immunohistochemical staining, sections were incubated with anti‐tyrosine‐hydroxylase (TH) (1:400; Abcam, catalog: ab75875) primary antibody for 48 h. The sections were then incubated with HRP‐conjugated anti‐rabbit IgG (1:50; Beyotime) secondary antibody. For immunofluorescence staining, tissue sections were permeabilized using 0.5% Triton X‐100 in PBS. The sections were incubated with anti‐CD3 antibody (1:100; Proteintech, catalog: 17617‐1‐AP) overnight. Sections were subsequently incubated with CY3‐labeled anti‐mouse secondary antibody (1:800; Abcam). Stained tissues were observed using a confocal fluorescence microscope (Leica).

In this study, we used human pancreatic adenocarcinoma cell line BxPC-3 which came from the American Type Culture Collection. The BxPC-3-DKK3 cell line was constructed according to our previous research [8 (link)]. RPMI 1640 medium supplemented with 10% fetal bovine serum were used for cells culture (37 °C, 5% CO₂). Besides, we used 10 μg/ml mitomycin (MilliporeSigma) pretrested the BxPC-3 cells for 2 h to inhibit expansion and ensure that the appropriate concentration of tumor-to-T cells was maintained. For co-cultivation, CD4⁺ T cells were first seeded into the lower chamber of a co-cultivation system for adherence, while BxPC-3 or BxPC-3-DKK3 cells were seeded into the upper chamber. After stimulated with 1 μg/ml anti-CD3 antibody (Proteintech, 17,617-1-AP) and 0.5 μg/ml anti-CD28 antibody (eBioscience, 16-0288-81) for 24 h, we started cells collection. Besides, sodium oxamate is the key enzyme of glycolysis which could enhance the hypoxic microenvironment by inhibiting glycolysis. BxPC-3 cells washed by PBS were pretreated with 50 mm sodium oxamate (Abcam) for 24 h and then washed before co-cultivation. Lactic acid (Tokyo Chemical Industry Co., Ltd.) was directly added into the co-culture. We used a three-gas incubator (MiniGalaxy A; RS Biotech) to create Hypoxic conditions (1% O₂) by injection of N₂ (1% O₂/94% N₂/5% CO₂ atmosphere) at 37 °C.