Pe rat anti mouse foxp3

Lamina propria cells (5 × 10⁶ cells) were fixed and permeabilized with the Foxp3/Transcription Factor Staining Buffer Set (Affymetryx eBioscience) and were stained with antibodies for (i) CD45 [Anti-Mouse CD45 APC-eFluor 780 (Affymetryx eBioscience)], F4/80 [PE/Cy7 Anti-Mouse F4/80 Antibody (BioLegend)], MHCII [Biotin Mouse Anti-Mouse I-A[d] (BD)], Ly6C [Anti-Mouse Ly-6C PerCP-Cyanine5.5 (Affymetryx eBioscience)], CD124 [PE Rat Anti-Mouse CD124 (BD)], CD206 [FITC Anti-Mouse CD206 (MMR) Antibody (BioLegend)], and IL-10 [APC Rat Anti-Mouse IL-10 (BD)]; (ii) CD45 [Anti-Mouse CD45 APC-eFluor 780 (Affymetryx eBioscience)], F4/80 [PE/Cy7 Anti-Mouse F4/80 Antibody (BioLegend)], MHCII [Biotin Mouse Anti-Mouse I-A[d] (BD)], Ly6C [Anti-Mouse Ly-6C PerCP-Cyanine5.5 (Affymetryx eBioscience)], CD3 [FITC Rat Anti-Mouse CD3 Molecular Complex (BD)], and IL-4 [PE Rat Anti-Mouse IL-4 (BD)]; and (iii) CD45 [Anti-Mouse CD45 APC-eFluor 780 (Affymetryx eBioscience)], CD4 [PE-Cy7 Rat Anti-Mouse CD4 (BD)], Foxp3 [PE Rat Anti-Mouse Foxp3 (BD)], and IL-10 [APC Rat Anti-Mouse IL-10 (BD)], as recommended by the manufacturers.
Data were acquired with the aid of the BD FACSCanto II flow cytometer (BD) and BD FACSDiva software (BD). The acquired data were analysed using FlowJo software, version 7.6.4 (TreeStar Inc.).

Splenocytes were collected from each mouse and the proportion of each T helper cell subset was analyzed: Th1 cells (CD4+ CD25+ T-bet+), Th2 cells (CD4+ CD25+ GATA-3+), Th17 cells (CD4+ CD25+ ROR-γt+), and Treg cells (CD4+ CD25+ Foxp3+). For analysis of the proportion of the T helper subsets, splenocytes were stained with antibodies to CD4 and CD25 (FITC rat anti-mouse CD4 and APC rat anti-mouse CD25; BD Biosciences, San Jose, CA, USA). Cells were washed, fixed, and intracellular staining was performed according to the manufacturer’s protocol for the Foxp3/transcription factor staining buffer set (eBioscience, San Diego, CA, USA) and intracellular antibodies (PE rat anti-mouse Foxp3; BD Biosciences, PE anti-RORγt, PE anti-T-bet, and PE anti-GATA-3; eBioscience). Fluorescence signals from 10,000 cells were counted, and flow cytometry analysis was carried out using a FACSAria flow cytometer (BD Biosciences). The proportion of CD138-positive cells was also determined (PE rat anti-mouse CD138, BD Biosciences).