β mercaptoethanol

Wheat flour (protein content 15%, w/w) and MBPs (protein content 92%, w/w) were purchased from a local market in Daqing, China. Additionally, β-Mercaptoethanol (β-ME) and sodium dodecyl sulfate (SDS) were obtained from Solarbio Co. (Beijing, China).

Synthetic Aβ fragment peptides 1–11, 12–28, 25–35 and 33–42 were purchased from Bachem Biotechnology (Bachem, Switherland). Aβ_1–42 was purchased from American Peptide Company (California, USA). Non-specific IgG (#A7028), HRP-conjugated secondary antibody (#A0216) and Propidium Iodide (PI) (#ST512) were obtained from Beyotime Biotechnology (Shanghai, China). Goat serum (#SL038), β-mercaptoethanol (#M8210) and Fluorescein Isothiocyanate Isomer I (FITC) (#F8070) were purchased from Solarbio Life Sciences (Beijing, China). Thioflavin T (ThT) (#T3516), Methyl Thiazolyl Triumvirate (MTT) (#M2128), Dimethyl Sulfoxide (DMSO) (#472301), All-trans-retinoic Acid (RA) (#R2625), Paraformaldehyde (#16005) and Diaminobenzidine (DAB) (#DA1010) were purchased from Sigma—Aldrich (USA). All other reagents were from Sigma-Aldrich, unless otherwise indicated.

Walnut meal was purchased from Yunnan Morre Gaden Biotechnology Development Co., Ltd. (Chuxiong, China). A BCA protein assay kit, Coomassie blue fast staining solution (R250), and SDS–PAGE protein sample loading buffer were purchased from Shanghai Beyotime Biotechnology Co., Ltd. (Shanghai, China). Alkaline protease (enzyme activity/titer: ≥200,000 U/g) and β-mercaptoethanol were purchased from Beijing Solarbio Technology Co., Ltd. (Beijing, China). SDS–PAGE precast gels (12%) were purchased from Wansheng Haotian Biotechnology Co., Ltd. (Shanghai, China). Chitosan oligosaccharides (molecular weight: ≤1000), phthaldialdehyde (OPA), sodium dodecyl sulfate (SDS), sodium tetraborate, CaCl₂, KCl, KH₂PO₄, NaHCO₃, NaCl, Mg₂Cl(H₂O)₆, and (NH₄)₂CO₃ were purchased from Shanghai Aladdin Bio-Chem Technology Co., Ltd. (Shanghai, China), and NaOH, HCl, methanol, acetic acid, and glutaraldehyde were purchased from Chengdu Chron Chemical Reagent Co., Ltd. (Chengdu China).

INS-1 cells (obtained from Bioleaf Biotech, Shanghai, China) were derived from a rat insulinoma. Cells were cultured in RPMI 1640 (GIBCO, California, USA) supplemented with 10% (v/v) fetal bovine serum (FBS) (10099141, GIBCO, Australia), 50 μM β-mercaptoethanol (Solarbio, Beijing, China), 10 mM HEPES (Solarbio), 2 mM L-glutamine (Solarbio), 1 mM Na pyruvate (Solarbio), 100 U/mL penicillin (Solarbio), and 100 μg/mL streptomycin (Solarbio), as described previously [14 (link)]. NG108-15 cells (obtained from China Center for Type Culture Collection (CCTCC), Wuhan, China) were cultured in Dulbecco's modified essential media (DMEM) (GIBCO) supplemented with 10% (v/v) FBS (GIBCO) and 1 × HAT (100 μM hypoxanthine, 0.4 μM aminopterin, and 16 μM thymidine) (H0262, Sigma). The cells were maintained at 37°C in a humidified atmosphere containing 95% air and 5% CO₂.

The determination method of GAD activity was assessed according to the method described by Khwanchai et al. [22 (link)], with some modifications. A 0.7 g sample of crushed maize was mixed with 3.2 mL of potassium phosphate buffer (0.05 M, pH 5.8). The potassium phosphate buffer contained 2 mM ethylenediaminetetraacetic acid (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China), 2 mM β–mercaptoethanol (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China), and 0.2 mM pyridoxal phosphate (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). The mixture was centrifuged at 1000× g for 20 min at 4 °C, and the resulting supernatant was used as the GAD extract. A 200 μL sample of GAD extract was mixed with 100 μL of 1% Glu (Sigma–Aldrich, St. Louis, MO, USA) and incubated for 2 h at 40 °C, pH 5.8, then the enzyme was inactivated at 90 °C for 10 min in a water bath and the GABA content was measured. The determination method of GABA content was described in Section 2.3.6. One unit of GAD activity was defined as the production of 1 μmol GABA from Glu per min at 40 °C.

The murine cell line DC2.4 (ATCC, USA, DCs, H-2^b) was incubated in RPMI-1640 medium with 1% Penicillin-Streptomycin (Solarbio, Beijing, China), 0.055 mM β-mercaptoethanol (Solarbio, Beijing, China), and 10% fetal bovine serum (Biological Industries, Beit haemek, Israel). The murine prostate cancer cell line (TRAMP-C1(ATCC) and RM1 (ATCC), H-2^b, obtained from C57BL/6) was incubated in RPMI-1640 medium with 1% Penicillin-Streptomycin, and 10% fetal bovine serum. All the experiments were performed using mycoplasma-free cells.

Fertilized eggs from Hy-Line chicken and Beijing You chicken (You chicken) were incubated in an incubator at 37.8 °C and 60% humidity. PGCs were isolated from the 10-day (E10) and 14-day (E14) female embryos of Hy-Line chicken and You chicken and cultured according to previously reported [20 (link),21 (link)]. Briefly, the isolated PGCs were cultured and amplified in the medium of knockout Dulbecco’s modified Eagle’s medium (KO-DMEM, Gibco, New York, NY, USA, 10829018) using chicken embryonic fibroblast cells as feeder cells at 39 °C in 5% CO₂ with saturated humidity. The KO-DMEM contained 10 ng/mL bFGF (Novoprotein, Suzhou, China, C044), 7.5% defined FBS (Solarbio, Beijing, China, P00032), 2.5% chicken serum (Solarbio, Beijing, China, S9080), 25 ng/mL human Activin-A (Novoprotein, Suzhou, China, C687), 1 × NEAA (Gibco, New York, NY, USA, 11140050), 1 × B-27 supplement (Gibco, New York, NY, USA, 17504044), 1 × GS nucleoside supplement (Millipore, Burlington, MA, USA, ES-008-D), 1 × antibiotic–antimycotic (Gibco, CA, USA, 15240062), 1 × Glu-taMAX (Gibco, New York, NY, USA, 35050061), 1.2 mM sodium pyruvate (Gibco, New York, NY, USA, 11360070), and 0.1 mM β-mercaptoethanol (Solarbio, Beijing, China, M8210).