Apc conjugated foxp3

Colon tissue was collected from acute colitis mice and cut into pieces in 1 mg/ml of collagenase IV; collagenase digestion was performed in collagenase solution for 45 minutes at 37 °C. The eluted cells were then passed through a 70-μl cell strainer and collected for 10 minutes at 1400 rpm at room. The cells were incubated in 10% donkey serum and Fc blocked (BD Pharmingen, San Jose, CA) for 20 minutes at 4 °C and then stained with fluorescent-conjugated antibodies. FITC-conjugated CD3, APC-conjugated CD25, PE-conjugated CD4 (OX-38), and APC-conjugated Foxp3 were purchased from BD Pharmingen (San Diego, CA) and used for the FACS analysis. The cells were analyzed with the FACSCaliber cytometer (BD Bioscience, San Diego, CA) and analyzed using FlowJo software (Tree Star, Ashland, OR).

Frequencies of T cells (CD3 + CD4 + , CD3 + CD8 + , CD4 + CD25 + Foxp3 +) and NK cells (CD3-NK1.1 + B220 +) were analyzed using the following antibodies (BD, Franklin Lakes, NJ, USA): APC-cy7-conjugated anti-CD3 (Clone SK7), PE-conjugated anti-CD4 (Clone RPA-T4), PerCP-conjugated anti-CD8 (Clone RPA-T8), PerCP 5.5-conjugated anti-CD25 (Clone PC61), APC-conjugated-Foxp3 (Clone 259D/C7), PE-conjugated anti-NK1.1 (Clone PK136), and PerCP-conjugated anti-B220 (Clone RA3-6B2). The estimation and analysis of T cells, NK cells, and cytokines were performed using Fortessa (BD Biosciences, San Jose, CA, USA) and FlowJo software (Tree Star, Ashland, OR, USA). T-cell proliferative capacities were assessed via flow cytometric analysis of carboxyfluorescein diacetate succinimidyl ester (Invitrogen, Carlsbad, CA, USA) in the mixed lymphocyte reaction (MLR) with irradiated (13 Gy) splenic DCs (1 × 10⁵ cells) as stimulators. For cytokine analyses, serial serum samples were collected and analyzed via cytometric Bead Array flex kits (BD Biosciences) containing IFN-γ, TNF-α, IL-2, IL-6, IL-10, and IL-17A.