Multiparametric Flow Cytometry Analysis

Single cell suspensions obtained from the intestinal mucosa or MLNs were stained and analyzed on FACSCanto II (Becton Dickinson, Franklin Lakes, NJ, USA) using FlowJo software v10.8.2 (TreeStar, Ashland, OR, USA). The following antibodies were used: anti-B220 (RA3-6B2; BioLegend), anti-CD38 (90; BioLegend), anti-CD45 (30-F11; BioLegend), anti-GL7 (GL7; BioLegend), anti-CD138 (281-2; BioLegend), anti-CD4 (GK1.5; BioLegend), anti-CXCR5 (L138D7, BioLegend), anti-PD-1 (29F.1A12, BioLegend), anti-Siglec-F (E50-2440, BD Biosciences, Franklin Lakes, NJ, USA), anti-CD11c (N418, BioLegend), anti-I-A/I-E (M5/114.15.2, BioLegend), anti-IgE (RME-1, BioLegend), anti-CD103 (W19396D, BioLegend), anti-CD11b (M1/70, BioLegend), anti-IL-7Rα (A7R34, BioLegend), anti-CCR6 (29-2L17, BioLegend), anti-TCRβ (H57-597, BioLegend), and anti-TCRγδ (UC7-13D5, BioLegend). Dead cells were gated out using a Zombie NIR Fixable Viability Kit (BioLegend). Before staining, Fc receptors were blocked with an anti-CD16/32 antibody (2.4G2, BioLegend). Negative controls consisted of isotype-matched, directly conjugated, nonspecific antibodies (BD Biosciences). Intracellular staining was performed using anti-Foxp3 antibody (FJK-16s; Thermo Fisher Scientific) and anti-RORγt antibody (Q31-378; BD Biosciences), as described previously [29 (link)].

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Kurihara S., Suzuki K., Yokota M., Ito T., Hayashi Y., Kikuchi R., Kageyama T., Meguro K., Tanaka S., Iwata A., Goto Y., Suto A, & Nakajima H. (2024). Eosinophils Contribute to Oral Tolerance via Induction of RORγt-Positive Antigen-Presenting Cells and RORγt-Positive Regulatory T Cells. Biomolecules, 14(1), 89.

Publication 2024

FACSCanto II anti-B220 (RA3-6B2; anti-CD45 (30-F11; anti-GL7 (GL7; anti-CD138 (281-2; anti-CD4 (GK1.5; anti-CXCR5 (L138D7, anti-PD-1 (29F.1A12, anti-Siglec-F (E50-2440, anti-CD11c (N418, anti-I-A/I-E (M5/114.15.2, anti-IgE (RME-1, anti-CD11b (M1/70, anti-IL-7Rα (A7R34, anti-CCR6 (29-2L17, anti-TCRβ (H57-597, Zombie NIR Fixable Viability Kit anti-CD16/32 antibody (2.4G2, nonspecific antibodies

Corresponding Organization : Chiba University

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Variable analysis

independent variables

Cell type (e.g., B220+, CD38+, GL7+, CD138+, CD4+, CXCR5+, PD-1+, Siglec-F+, CD11c+, I-A/I-E+, IgE+, CD103+, CD11b+, IL-7Rα+, CCR6+, TCRβ+, TCRγδ+)

dependent variables

Frequency or expression levels of the cell surface markers and intracellular markers (Foxp3, RORγt)

control variables

Fixation and permeabilization conditions for intracellular staining
Gating strategy (e.g., exclusion of dead cells, Fc receptor blocking)

positive controls

Isotype-matched, directly conjugated, nonspecific antibodies

negative controls

Isotype-matched, directly conjugated, nonspecific antibodies

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