Pe rat anti mouse cd4

AMP-IBP5 (AVYLPNCDRKGFYKRKQCKPSR-NH₂; molecular weight: 2655.1) was obtained from the Peptide Institute (Osaka, Japan) and was dissolved in acetic acid (0.01%). IgG isotype control and antibodies specific for phosphorylated and unphosphorylated aPKCζ and Rac1 were purchased from Cell Signaling Technology (Beverly, MA, USA). A rabbit monoclonal antibody specific for claudin-1 was purchased from Cell Signaling Technology. Mouse monoclonal antibodies specific for claudin-4, occludin, and ZO-1 were obtained from Invitrogen (Carlsbad, CA, USA). A rabbit polyclonal antibody specific for claudin-7 was purchased from Invitrogen. The aPKCζ inhibitor GF 109203X was obtained from Enzo Life Sciences (Farmingdale, NY, USA). The Rac1 inhibitor NSC23766 was obtained from Calbiochem (La Jolla, CA, USA). Enzyme-linked immunosorbent assay (ELISA) kits were obtained from R&D Systems (Minneapolis, MN, USA). The EZ-link^TM Sulfo-NHS-LC-Biotin tracer was purchased from Thermo Scientific (Waltham, MA, USA). Recombinant human and mouse LRPAP were obtained from R&D Systems. Toluidine blue solution (0.05%, pH 4.1) was purchased from Muto Pure Chemicals Co. Ltd. (Tokyo, Japan). Recombinant human IL-4 and IL-13 were obtained from BioLegend (San Diego, CA, USA). A mouse monoclonal antibody specific for CD4 (PE rat anti-mouse CD4) was purchased from BD Bioscience (San Jose, CA, USA).

Using a well-established protocol [3 (link)], splenic CD4⁺CD45RB^high T cells from C57BL/6 WT mice were adoptively transferred into same-sex 8–12-week-old Rag1^tm1Mom mice. First, the spleens from C57BL/6 WT mice were removed and placed in FACS buffer (PBS, fetal bovine serum, and sodium azide). Then, the spleens were homogenized by rubbing between glass slides until a single-cell suspension was created. The single-cell suspension was passed through a 70µm cell strainer. Cells were enriched by negative isolation using Dynabeads Untouched Mouse CD4 Cell Kit (Life Technologies AS, Carksbad, CA, USA) according to the manufacturer’s instructions. CD4 cells were fluorescently labeled with fluorescein isothiocyanate (FITC) rat anti-mouse CD45RB (Thermo Fischer Scientific, Waltham, MA, USA) and phycoerythrin (PE) rat anti-mouse CD4 (BD Biosciences, Franklin Lakes, NJ, USA). CD4+ T cells were sorted using a Becton-Dickenson Influx cell sorter by double gating on CD4⁺ singlet cells and the brightest 40% CD45RB^high cells. Cells were sorted into tubes containing post-sorting buffer (PBS, EDTA, FBS) to stabilize the post-sorted T cells. In total, 5 × 10⁵ CD4⁺CD45RB^high T cells were then injected intraperitoneally into recipient Rag1^tm1Mom mice of the same sex as the T cell donors.

At 21 and 42 days of age, splenic samples of eight mice in each group were taken to determine the percentages of CD3⁺, CD4⁺, CD8⁺ T lymphocyte and CD 19⁺ B lymphocyte by FCM.
Each spleen was cut into pieces and then filtered through nylon gauze as splenic single-cell suspension. The suspension was centrifuged at 200 × g for 5 min. The supernatant was discarded and lymphocytes were collected. The cell concentration was determined by using the normal counting method of blood cells and then diluted to 1.0 × 10⁶ cells/mL with phosphate-buffered saline (PBS). A total of 100 μL cell suspensions was transferred to another centrifuge tube. The cells were respectively stained with 10 μL hamster anti-mouse CD3e-FITC (BD, Cat No: 553062), rat anti-mouse CD4-PE (BD, Cat No: 557308), rat anti-mouse CD8a-PerCP (BD, Cat No: 553036) and FITC anti-mouse CD19⁺(BD, Cat No: 553785) for 30 min at RT, and then 2 mL PBS was added and centrifuged at 200 × g for 5 min. The supernatant was discarded. Cells were resuspended in 0.5 mL PBS and determined by BD FACS Calibur flow cytometer.

Splenocytes were stained with Mouse Anti-Mouse NK 1.1- APC (BD Biosciences, USA), Rat Anti-Mouse CD3-PE (BD Biosciences, USA), Rat Anti-Mouse CD4-PE (BD Biosciences, USA), and Rat Anti-Mouse CD8-APC (BD Biosciences) according to the manufacturer’s instructions. After staining, cells were analyzed by flow cytometry (Novocyte Flow Cytometer, ACEA Biosciences, USA). The positivity of CD8, CD4, and CD3 was determined by comparison with the defined cutoff values obtained with unstained control cells as previously described [18 (link)].