Apc anti foxp3 fjk 16s

Cells derived from Peyer’s patches (PPs) and cervical lymph nodes (CLNs) were stained with specific surface markers (CD3, CD4) for flow cytometry using fluorescence-conjugated anti-mouse monoclonal antibodies (Abs); i. e., PE-Cy7-anti-CD3 (145-2C11) and Pacific blue-anti-CD4 (RM4-5) Abs (BD Biosciences, NJ, USA). To analyze the cells expressing cytokines IFN-γ, IL-4 and IL-17, intracellular staining was performed using Intracellular Fixation & Permeabilization Buffer (eBioscience, CA, USA) and PerCP-Cy5.5-anti-IFN-γ (XMG1.2), PE-anti-IL-4 (11B11) and APC-anti-IL-17A (17B7) Abs (eBioscience, CA, USA) after stimulation for 16 h with a Cell Stimulation Cocktail (Invitrogen, MD, USA) as described in by the manufacturer. Furthermore, transcription factor FoxP3 was stained by intracellular staining using APC-anti-FoxP3 (FJK-16s) Ab according to the protocol included with the Foxp3/Transcription Factor Staining Buffer Set (eBioscience, CA, USA). The cells were analyzed using a FACS Canto II system (BD Biosciences). In each experiment, specimens were analyzed for singlet events with doublet discrimination. All data were examined by FACSDiva software (BD Biosciences, NJ, and USA) and Flowjo software (Tree Star, OR, USA).

To obtain single-cell suspensions, lymphocytes were isolated from the thymus, spleen, inguinal lymph nodes, lungs, and livers as described before^{35 (link)}. The prepared cells were stained with the following antibodies: anti-CD4-BV605 (RM4–5, BioLegend), anti-PD-1-BV421 (RMP1–13, BioLegend), anti-CD2-PE-Cy7 (RM2–5, BioLegend), anti-CD23-PerCP-Cy5.5 (B3B4, BioLegend), anti-CD21/35-BV421 (7E9, BioLegend), anti-CD43-PE (1B11, BioLegend), anti-IgM-APC (RMM-1, BioLegend), anti-IgD-BV605 (11-26c.2a, BioLegend), anti-CD8-PerCP-Cy5.5 (53-6.7, eBioscience), anti-CD25-FITC (PC61.5, eBioscience), anti-CD3-PE-Cy7 (145-2C11, BD Biosciences), anti-CD44-V450 (IM7, BD Biosciences), anti-CD45R/B220-FITC (RA3-6B2, BD Biosciences), anti-CD62L-FITC (MEL-14, BD Biosciences), anti-CD69-PE (H1.2F3, BD Biosciences), and anti-TCR-β-FITC (H57-597, BD Biosciences). For the staining of transcription factor FoxP3, the cells were stained using anti-FoxP3-APC (FJK-16s, eBioscience) after permeabilization/fixation step. In all experiments, cells were labelled with the Live/Dead fixable Stain Kit (eBioscience) to discriminate the live and dead cells. The data were acquired by FACS CANTO II (BD Biosciences) and analysed by FlowJo software (Tree Star).

Cell suspensions were generated from spleens and PALNs by gentle homogenization in RPMI 1640 (Gibco^TM, Life Technologies, Invitrogen, Australia Pty. Ltd.). Erythrocytes were lysed in splenocyte suspensions using red blood cell lysis buffer (155 mM NH₄Cl, 10 mM KHCO₃ and 0.1 mM EDTA). Dead cell exclusion was enabled by addition of BD Horizon™ Fixable Viability Stain 620 (Becton Dickinson (BD), Franklin Lakes, NJ). 10⁶ cells were stained in PBS (0.05% sodium azide, 0.1% BSA (Sigma). Fc receptors were blocked with anti-CD16/CD32 Fc Block™ (Mouse Fc Block™; BD) before surface staining with antibodies; anti-CD4 APC-Cy7 (GK1.5, BD), anti-CD8α PE-Cy7 (53–6.7, BD) and Nrp1 BV421 (3E12, Biolegend, San Diego, CA). Intracellular staining was performed using Foxp3 Staining Buffer Set (eBioscience, San Diego, CA), as per the manufacturer’s instructions, and anti-Foxp3 APC (FJK-16s, eBioscience) antibody. Data were acquired on a BD FACSCantoII. All data were analysed using FlowJo software (TreeStar, Inc., Ashland, OR).