Fitc labeled anti mouse cd3

Facial lymph nodes (FLN) and axillary lymph nodes (ALN) were collected and mechanically disaggregated. A single-cell suspension from the FLN and the ALN of each mouse was obtained by gently passing the collected tissue through a tissue strainer with PBS with 2% FCS (FACS buffer). Cell suspensions were subjected to red blood cells lysis (Tris-ammonium chloride, BD PharMingen, Frankiln Lakes, NJ, USA) flowed by counting on a hemacytometer. The viability of cells was assessed by trypan blue exclusion. Cell suspensions were pre-incubated with anti-mouse CD32/CD16 monoclonal antibody (Fc block) for 30 min at 4 °C. Cells were incubated with the antibody mixes for 30 min at 4 °C and washed with FACS buffer. The following antibodies from BD Biosciences were used: FITC-labeled anti-mouse MHC-II, FITC-labeled anti-mouse CD3, biotinylated anti-mouse CD4, FITC-labeled anti-mouse CD25, and PE-labeled anti-mouse CD11c antibodies. Streptavidin-PerCP was used as a second-step reagent. Flow cytometry was performed using a BD FACSCalibur^TM flow cytometer (BD Biosciences, Frankilin Lakes, NJ, USA) and data were analyzed using FlowJo software (TreeStar, BD Biosciences, NJ, USA).

Ilium Payer's patches and spleens were collected and mechanically disaggregated. A single-cell suspension from the Peyer's patches or spleens of each mouse was obtained by gently passing the collected tissue through a tissue strainer with PBS supplemented with 2% FCS (FACS buffer). Cell suspensions were subjected to red blood cells lysis (Tris-ammonium chloride, BD PharMingen) and counted on a hemocytometer. Trypan blue exclusion method was used to assess viability of cells. Cell suspensions were pre-incubated with anti-mouse CD32/CD16 monoclonal antibody (Fc block) for 30 min at 4°C. Cells were incubated with the antibody mixes for 30 min at 4°C and washed with FACS buffer. The following antibodies from BD Biosciences were used: PE-labeled anti-mouse CD24, biotinylated anti-mouse B220, PE-labeled anti-mouse CD45, biotinylated anti-mouse CD45, FITC-labeled anti-mouse CD3, PE-labeled anti-mouse CD8, and biotinylated anti-mouse CD4 antibodies. Streptavidin-PerCP was used as a second-step reagent. Flow cytometry was performed using a BD FACSCaliburTM flow cytometer (BD Biosciences) and data were analyzed using FlowJo software (TreeStar).

Spleen and kidney tissue were harvested from mice at given time points and crushed in mesh bags to obtain single cell suspensions, in which RBC were lysed with hypotonic erythrocyte lysis buffer (TIANGEN Biotech, Beijing, China). Thereafter all single cells were re-suspended in staining buffer (BD Bioscience, San Diego, CA, USA). The following monoclonal antibodies (1 mg/ml; BD PharMingen) were used to identify NKT cells: FITC-labeled anti-mouse CD3 (Clone: 17A2) and APC-labeled anti-mouse NK1.1 (Clone: PK136). CXCR3 expression was determined by PE-labeled anti-mouse CXCR3 (CD183) (Clone: CXCR3-173). Flow cytometry data were acquired using BD FACS Aria II (BD Biosciences) and analyzed with FlowJo software 6.0 (Tree Star Inc., Ashland, OR, USA).