Apc cy7 conjugated cd4

For human B cell staining, cells were stained with PE-Cy7-conjugated anti-CD19 (BioLegend, #302216), PE-conjugated CD27 (BioLegend, #302808), BV421-conjugated IgD (BioLegend, #348226), BV421-conjugated anti-CD138 (BioLegend, #356516), and APC-conjugated anti-IgG antibodies (BioLegend, #304136). Alternately, cells were permeabilized and stained with primary antibodies against ORAI1, ORAI2, and STIM2 at 37°C for 30 min. Cells were then washed with phosphate-buffered saline (PBS) and incubated with Alexa Fluor^® 488 Goat antirabbit IgG (Thermo Fisher, #A-11008) secondary antibodies at room temperature for 30 min. For mouse cell staining, cells were stained with APC-conjugated B220 (BioLegend, #318326), PE-Cy7-conjugated CD38 (BioLegend, #102718), BV421-conjugated CD138 (BioLegend, #142523), PE-conjugated PD-1 (BioLegend, #135205), APC-conjugated CXCR5 (BioLegend, #145506), PE-conjugated CD95 (BioLegend, #152608), fluorescein isothiocyanate (FITC)-conjugated GL-7 (BioLegend, #144612), APC-Cy7-conjugated-CD4 (BioLegend, #100526), BV510-conjugated-CD3 (BioLegend, #100234), Percp-cy5.5-conjugated-CD45 (BioLegend, #103132), APC-conjugated-CD8 (BioLegend, #100712), PE-Cy7-conjugated-CD19 (BioLegend, #506921) and BV605-conjugated-CD11b (BioLegend, #101257). Samples were analyzed using a BD LSR Fortessa (BD Bioscience).

C57BL/6 mice were purchased from Chubu Kagaku Shizai (Nagoya, Japan). All the mice were handled in accordance with the Guidelines for Animal Experiments, Gifu University (Gifu, Japan). Mice are sacrificed by cervical dislocation. The APC-Cy7-conjugated CD4, PE-Cy-7-conjugated CD8 antibodies, PE-conjugated CD80 antibody, the purified anti-mouse CD16/32, the APC-Cy7-conjugated rat anti-mouse CD45, APC-Cy7-conjugated TER-119, and PE-conjugated anti-mouse EpCAM (G8.8) were obtained from Biolegend (San Diego, CA). The rabbit anti-mouse keratin-5 antibody was purchased from Covance (Berkeley, CA). The biotinylated anti-mouse keratin-8 antibody was from PROGEN (Heidelberg, Germany). The biotinylated UEA-1 was obtained from VECTOR Laboratories (Burlingame, CA). Further, 7-aminoactinomycin D was purchased from Wako (Tokyo, Japan).

To determine cell types infiltrating the lung and leukocytes present in peripheral blood, single cell suspensions from the tissues mentioned above were prepared, as described previously.⁴² Briefly, lung tissue was homogenized by Dounce homogenizers, filtered with a 100 µm filter, and red blood cells were lysed for 2 min at 37 °C (Pharmlyse). Single cell populations were blocked by initial incubation with Fc Block (BD, 553141) for 15 min at 4 °C. Cell populations were incubated in the dark with antibodies to cell surface markers for 1 h at 4 °C. Neutrophil populations were identified using: PE-conjugated GR-1 (BD, 553128,1:500), Alexa Fluor 700-conjugated CD11b (Biolegend, 101222, 1:500). Neutrophils were classified as CD11b⁺Gr-1^hi single, live cells. TRM populations were determined using: APC-Cy7-conjugated CD4 (Biolegend, 100526, 1:500), BB515-conjugated CD44 (BD, 564587, 1:500), APC-conjugated CD62L (BD, 553152, 1:500), and BV421-conjugated CD69 (BD, 562920, 1:500). The TRM gating strategy was adapted from Wilk et al., shown in Supplementary Fig. 1.^{21 (link)}