Igd pe

Mice aged 6 to 8 weeks old were immunized with sheep red blood cells (SRBC) and spleens were harvested 9 days later. Spleens from 1-year-old mice were also collected to analyze T_FH cells and GC formation. The tissues were frozen in OCT reagent and sectioned into 7-µm slices. The frozen sections were stained with anti-PNA-FITC (Sigma-Aldrich, St. Louis, MO), IgD-PE, and CD4-APC (eBioscience) overnight at 4°C. After washing, Anti-Fade reagent (Invitrogen, Life Technologies, Carlsbad, CA) was added to the slides, which were visualized using a Leica DM IRE2 confocal microscope.

Directly conjugated antibodies were acquired from the following: (1) BD Biosciences: CD3-H7APC, CXCR5-Alexa488 (RF8B2), CCR7-Alexa700, IgG-APC, IFN-γ-Alexa700 and IL-21-Alexa647 (3A3-N2.1) (2) Beckman Coulter: CD45RO-ECD and CD27-PC5 (3) Biolegend: CCR7-BV421, CCR6-PE (TG7/CCR6), CCR6-Alexa647 (TG7/CCR6), CD20-BV570, CD150-PE, IL-2-BV605, IL-17a-Cy5.5PerCP and CD154-Cy5PE (4) Invitrogen: CD4-Cy5.5PE, CD27-QD655, CD27-QD605 and CD19-PacBlue (5) Southern Biotech: IgD-FITC and IgD-PE (6) eBioscience: cmaf-eFluor660 (sym0F1), CXCR5-PerCP-efluor710 (MU5UBEE). Biotinylated anti-PD-1 was from R&D and streptavidin-Cy7PE (or QD655) was from Molecular Probes. The following antibodies were conjugated in our lab: CD19-QD705 and CD57-QD565. Quantum dots and Aqua amine viability dye were obtained from Invitrogen.

For GC formation analysis, the spleens from six- to eight-week-old sheep red blood cell (SRBC)-immunized mice were isolated 7 days after immunization and frozen in OCT compound. Tissues were sectioned to a 7-μm thickness, fixed in acetone at −20 °C, washed with PBS, and blocked with 0.1% BSA containing PBS for 30 min at room temperature. Tissues were stained with anti-PNA-FITC (Sigma-Aldrich, St. Louis, MO, USA), IgD-PE, and CD4-APC (eBioscience) antibodies diluted in blocking solution overnight at 4 °C. After three washes, tissues were incubated with ProLong Gold anti-fade reagent (Invitrogen, Life Technologies, Carlsbad, CA, USA) for 30 min at 4 °C and images were obtained using a Leica DM IRE2 confocal microscope.

Single cell suspensions were washed with PBS and 1.0 × 10⁶ cells/tube were FC blocked (eBioscience) and stained extracellularly for 30 min at 4 °C. Cells were then washed and prepared for intracellular staining using the FoxP3 Intracellular Staining Kit (R and D systems). Intracellular staining was performed at 4 °C for 60 min. Antibodies CD79A-APC, CD27-FITC, IgD-PE, CD4-APC, CD45RO-PE, CD8-FITC, and CD3-APC were purchased from eBioscience.

Spleens of immunized mice were harvested at indicated time points and single cell suspensions were obtained by passing through 70 μm cell strainer (Greiner Bio-ONE, cat. 542070). Single cells were suspended in FACS buffer (2% FBS in PBS) and incubated with antibody at 4°C. To distinguish the Qβ-specific B cells in germinal center, cells were firstly stained with Qβ-AlexaFluoro 488 and PNAbio (Vector Laboratories, cat. B-1075), and then Fc-receptors were blocked with anti-mouse CD16/CD32 (BD Bioscience, cat. 553142). Finally, streptavidin-APC Cy7 (BD Bioscience, cat. 554063) and anti-mouse CD38-PerCP Cy5.5 (Biolegend, cat. 102722) were applied to determine germinal center cells, anti-mouse B220-PE Cy7(BD Bioscience, 552772) for B cells, anti-mouse IgM-PE (Jackson ImmunoResearch, cat. 115-116-075), IgD-PE (eBioscience, cat. 12-5993-83), CD4-PE (BD Bioscience, cat. 553653), CD8-PE (BD Bioscience, cat. 553032), CD11b-PE (BD Biosience, cat. 553311), CD11c-PE (BD Biosience, cat. 553802), GR1-PE (BD Biosience, cat. 553128) to exclude other cell types (CD4, CD8: T cells; CD11b: monocytes and macrophages; CD11c: dendritic cells; GR1: neutrophils) and immature B (IgM⁺IgD⁺) cells.