Mouse th1 th2 th17 cba kit

Cytokine concentrations in supernatants were determined using a mouse Th1/Th2/Th17 CBA kit (BD Biosciences), which allowed for the simultaneous detection of IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ, and IL-17A. Aliquoted samples were thawed, and CBA analysis was performed according to the manufacturer’s protocol. Briefly, beads coated with capture antibodies were mixed, and 50 μl of the capture bead mixture was added to 50 μl of sample. To these sample bead complexes, 50 μl of phycoerythrin (PE)-conjugated detection antibody was added, and this mixture was incubated for 3 h in the dark at room temperature. Samples were washed with 1 ml of wash buffer at 1100 rpm for 5 minutes, and the pellets were resuspended in 300 ml of wash buffer. Cytokine standards were serially diluted to facilitate the construction of calibration curves necessary for determining protein concentrations of test samples. Flow cytometric analysis was performed on a BD FACSCanto II (BD Immunocytometry Systems, Erembodegem, Belgium) with BD FACSDIVA version 6 software, and data were analyzed with FCS Express version 3 software (De Novo Software, Glendale, CA, USA).

Single cell suspensions of splenocytes were prepared as described above, counted using a hemocytometer in trypan blue (Sigma, AUS) and adjusted to 4 × 10⁶ cells/mL. Splenocytes were stimulated with heat-killed 2031 or medium as a negative control. After 72-hour stimulation at 37°C with 5% CO₂, the cells were centrifuged at 300g for 10 min, and the supernatant was collected. The supernatants were stored at −80°C. Cytokines in the supernatant were subsequently measured using a mouse Th1/Th2/Th17 CBA kit (BD, AUS) according to the manufacturer’s instructions. Samples were acquired using a BD LSR Fortessa cytometer, and data were analyzed using FCAP Array v3.1 (BD).

Luteolin and DTT were obtained from Aladdin (China). 1,4-butanediol diacrylate (BDD) was purchased from TCI (Shanghai, China). Dextran sulfate sodium (DSS, 35000 ​Da) was purchased from MP Biomedical (USA). Mouse Th1/Th2/Th17 CBA Kit, Cytofix/CytopermSoln Kit, Leuko Act Cktl with GolgiPlug, Transcription Factor Buffer Set, Fixable Viability Stain 780, FITC-Anti-CD4 antibody, BV605-Anti-IL-17A antibody, APC-Anti–INF–γ antibody and PE-Anti-IL-4 antibody were purchased from BD Biosciences (San Diego, USA). PE-Anti-FOXP3 antibody was purchased from eBiosciences (San Diego, CA). Myeloperoxidase (MPO) kit, glutathione (GSH) kit and ROS kit were supplied by Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The BCA protein determination kit and Reactive Oxygen Species Assay Kit were purchased from Beyotime Biotechnology (China). Mouse Beta Actin antibody, Mouse Occludin antibody and Mouse ZO-1 antibody for western blotting were purchased from Proteintech (China). SYBR Premix Ex Taq™, Trizol reagent and PrimeScript™ RT 131 Master Mix were obtained from TaKaRa Bio (China). All other reagents are commercially available and can be used directly. All the solvents used were of analytical grade and were procured from Sinopharm (China).

The spleens of the mice were removed under aseptic conditions after euthanasia and ground. The mouse spleen cells were diluted to 4×10⁶/mL using 1640 medium containing 10% FBS, and 50 μL of the cell suspension was added to a 96-well cell culture plate. Thereafter, the peptide library or antigen diluent was added for stimulation. The 96-well plates were incubated in a cell culture incubator at 37°C for 3 days. After stimulation, the supernatant was centrifuged at 500 g for 5 min and collected. Cytokine secretions of the splenocytes were analyzed using mouse Th1/Th2/Th17 CBA Kit (BD Pharmingen, USA) following the previously described procedure. Supernatants were collected and aliquoted for the cytokine assay. Captured bead reagents were applied to the mixture of the 50 mL sample and PE-detection antibody. The reaction lasted 2 h in the dark at room temperature. All unbound antibodies were removed by the addition of 1 mL of wash buffer before centrifugation. Captured beads were then resuspended with 300 μL wash buffer and analyzed using FACS (BD). Six standard curves were acquired for each experiment using four-parameter linear fitting for the concentrations of serially diluted standards ranging from 0-5000 pg/mL.

Concentrations of IFN-γ in serum from acute hepatitis mice/control mice were measured with a cytometric bead array kit (Mouse Th1/Th2/Th17 CBA kit, BD Biosciences) and analyzed using a FACS Verse flow cytometer with CBA software (BD Biosciences).

Serum concentrations of IFN-γ, TNF-α, IL-4 and IL-17A from Il2ra^−/−Tg and control mice were measured with a cytometric bead array kit (Mouse Th1/Th2/Th17 CBA kit, BD Biosciences), using a FACSVerse flow cytometer with CBA software (BD Biosciences).

The level of cytokines in cell culture supernatants was determined by flow cytometry using a commercial cytometry bid array Mouse Th1/Th2/Th17 CBA Kit (BD Biosciences, San Diego, CA, USA, cat. no. 560485). The sample preparation was carried out following the manufacturer’s protocol. Data collection was performed on a Beckman MoFlo XDP flow cytometer (Beckman Coulter, Miami, FL, USA) with Summit V5.2.0.7477 software installed. The following device configuration was used in the experiment: blue, green, and red lasers with wavelengths of 488 nm, 561 nm, and 628 nm, respectively. The Fl-8 (580/23 nm)/Fl-10 (670/30 nm) channels were used to detect the signal from particles carrying PE-conjugated antibodies. Processing and analysis of the results were carried out using the FCAP Array Software v3.0—BD Biosciences. Mean and standard deviation from different replicas were used to determine the concentration of the cytokines.