Pe cy7 conjugated anti mouse cd3

For the analyses of hematopoietic cells, a hypotonic lysis was performed to remove erythrocytes. 50 μl of blood was lysed using 500 μl of ACK (Ammonium-Chloride-Potassium) Lysing Buffer (Lonza, Walkersville) 10 min at room temperature. After the erythrocytes lysis, two washes with PBS were performed prior the incubation with the antibodies for 30 min at 4°C. Cells were stained with the following fluorochrome-coupled antibodies: Allophycocyanin (APC)-conjugated anti-mouse CD11b (1:300; cn.17–0112 eBioscience, USA) to label myeloid cells, phycoerythrin (PE)-conjugated anti-mouse B220 (1:100; cn.12–0452, eBioscience, USA) for B lymphocytes and phycoerythrin/cyanine (PE/Cy7)-conjugated anti-mouse CD3, 1:100; cn.100320, BioLegend, USA) for T lymphocytes. Immunofluorescence of labeled cells was measured using a BD LSR II flow cytometer. Dead cells and debris were excluded by measurements of forward- versus side-scattered light and DAPI (4′,6-diamino-2-phenylindole) (Sigma) staining. Gates for the respective antibodies used were established with isotype controls and positive cell subset controls. Data analysis was carried out using FACSDiva version 6.2 software (BD biosciences).

C57BL/6 and Rag1^−/− mice were purchased from Jackson Laboratory. All mice were kept in sterilized, ventilated cages under SPF conditions and fed either a control diet or exposed to AR via the diet. Mice were allowed to acclimatize for 7 days prior to the start of experiments. Colitis was induced in Rag1^−/− mice by adoptive transfer of FACS-sorted CD4⁺CD45RB^hi T cells. Naïve CD4⁺ T cells were isolated from splenocytes of C57BL/6 mice by EasySep^TM Mouse Naïve CD4⁺ T cell Isolation Kit (StemCell Technology, Vancouver, Canada). Naïve CD4⁺ T cells were labeled with PE-cy7-conjugated anti-mouse CD3 (BioLegend; 1:100), APC-conjugated anti-mouse CD4 (BD Biosciences; 1:100), and FITC-conjugated anti-mouse CD45 (BD Biosciences; 1:100). CD4⁺CD45RB^hi T cells were sorted using FACS Aria II flow cytometer (BD Biosciences; FACSDiva v 6.1.2). Cell viability was assessed using Trypan blue assay prior to injection. Recipients were intraperitoneally (i.p.) injected with 5 × 10⁵ cells of sorted T cells. Samples from all mice were kept at −80 °C.

In order to evaluate the phenotype of different immune cell populations, cells derived from the spleen or tumor of vehicle- or AZD1480-treated mice were stained with the following antibodies: phycoerythrin (PE)-Cy7-conjugated anti-mouse CD3 (BioLegend), Alexa Fluor 700 (AF700)-conjugated anti-mouse CD4 (BD Biosciences), AF647-conjugated anti-mouse CD8 (BioLegend), Horizon V450-conjugated anti-mouse CD45 (BD Biosciences), peridinin chlorophyll protein (PerCP)-Cy5.5-conjugated anti-mouse CD4 (BD Biosciences), PE-conjugated anti-mouse CD25 (eBioscience), AF700-conjugated anti-mouse CD127 (eBioscience), AF647-conjugated anti-mouse CD11c (BioLegend), PE-conjugated anti-mouse CD11b (BD Biosciences), fluorescein isothiocyanate (FITC)-conjugated anti-mouse CD86 (BD Biosciences), allophycocyanin (APC)-H7-conjugated anti-mouse CD80 (BD Biosciences), FITC-conjugated anti-mouse CD11b (BD Biosciences), AF647-conjugated anti-mouse Ly6G (BioLegend) and PECy7-conjugated anti-mouse Ly6C (BioLegend).