Pe conjugated rat antimouse b220 clone ra3 6b2

A piece of the salivary glands obtained from 24-week-old mice was embedded in OCT compound (Sakura Finetek, Torrance, CA, USA) and frozen in liquid nitrogen. Sections of 10-μm thickness were blocked with 10% horse serum in PBS. The sections were stained with mouse antihuman CD3-ζ mAb clone 6B10.2 (catalog No. sc-1239, 1:75 dilution, Santa Cruz Biotechnology Inc, Dallas, TX, USA) and PE-conjugated rat antimouse B220 clone RA3-6B2 (catalog No. 553090, 1:75 dilution, BD Bioscience, San Jose, CA, USA) followed by goat antimouse IgG (H + L) Alexa 488 catalog No. A28175 (1:1000 dilution, Thermo Scientific, Waltham, MA, USA). Nuclei were stained with DAPI. Immunofluorescence images were captured under a confocal microscope (Leica, Wetzlar, Germany).

To isolate mononuclear cells from NALT, NALT was first obtained from the upper jaw of the mice. NALT cells were isolated by gently rubbing the NALT sample with a needle under a stereoscopic microscope. After washing with PBS, the collected cells were treated with anti-mouse CD16/32 (clone 93; BioLegend, San Diego, California, USA) for 15 min at room temperature. After washing with PBS containing 2% newborn calf serum, the cells were stained with fluorescein isothiocyanate-conjugated hamster anti-mouse CD3ε (clone 145-2C11, BD Biosciences, San Diego, California, USA), phycoerythrin (PE)-conjugated rat anti-mouse B220 (clone RA3-6B2, BD Biosciences), PE-conjugated rat anti-mouse PD-1 (clone 29F1.A12, BioLegend), Alexa Fluor 647-conjugated rat anti-mouse GL7 (clone GL7, BioLegend), PE-Cy7-conjugated rat anti-mouse CD4 (clone RM4-5, BD Biosciences), PE-Cy7-conjugated Armenian hamster anti-mouse CD11c (clone N418, BioLegend), APC-Cy7-conjugated rat anti-mouse CD8α (clone 53-6.7, BD Biosciences), and Brilliant Violet 421-conjugated rat anti-mouse CD45 (clone 30-F11, BioLegend) for 30 min at 4 °C. After washing with PBS containing 2% newborn calf serum, cells were treated with 7-Amino-Actinomycin D (BioLegend) for 10 min at 4 °C and analyzed by means of flow cytometry (MACSQuant) (Miltenyi Biotec, Auburn, California, USA).

A week after the last immunization, the NALT and nasal passage were harvested from the upper jaw of the mice. Mononuclear cells from NALT were obtained by scraping the NALT or nasal passage tissue. After treatment with ACK lysis buffer to remove red blood cells, the cells were suspended in a staining buffer (PBS containing 2% heat-inactivated fetal bovine serum and 0.1% sodium azide) and then treated with anti-mouse CD16/32 antibody (clone 93; BioLegend) for 30 min at 4 °C to block Fc receptors. After washing with staining buffer, the cells were stained with phycoerythrin (PE)-conjugated rat anti-mouse B220 (clone RA3-6B2; BD Bioscience, San Diego, CA, USA), fluorescein isothiocyanate (FITC)-conjugated rat anti-mouse IgA (clone C10-3; BD Bioscience), or PE-conjugated rat anti-mouse CD138 (clone 281-2; BD Bioscience) for 30 min at 4 °C. After washing with staining buffer, the cells were incubated with 7-amino-actinomycin D (7-AAD; BioLegend) for 10 min at 4 °C and then analyzed using a FACSCalibur instrument (BD Bioscience).