Ebio fix perm kit

For ABCs detecting, PBMCs/B cells to be detected were incubation with FcR blocking reagent (Miltenyi Biotec) for 10 minutes at 4 °C, then stained with BV421 anti-CD11c (BioLegend) and percp-Cy5.5 anti-CD19 (BioLegend) at 4 °C for 30 minutes. For intracellular staining, cells were surface stained, fixed using eBio fix/perm kit (eBioscience), washed and stained with PE-Cyanine7 anti-T-bet (BioLegend) in 1 × eBio fix/perm buffer for 1 hour at 4 °C. Mouse IgG1 kappa Isotype Control, PE-Cyanine7 (eBioscience) was used as isotype control for T-bet staining.
For RNASE2 detecting, PBMCs to be detected were incubation with FcR blocking reagent (Miltenyi Biotec) for 10 minutes at 4 °C, then stained with APC anti-CD14 and viability dye efour 506 (Invitrogen) at 4 °C for 30 minutes. For intracellular staining, cells were surface stained, fixed with fixation/permeabilization solution (Cytofix/Cytoperm kit, BD), washed and stained with RNASE2 antibody (Invitrogen) in 1× Perm/Wash buffer for 1 hour at 4 °C, then stained with AlexaFlour 488 Goat anti-rabbit IgG (FCMACS). Cells stained with CD14 and AlexaFlour 488 Goat anti-rabbit IgG were used as isotype control for RNASE2 staining.
Then Cells were analyzed on a BD FACSAria III flow cytometer (BD Biosciences) using FACSDiva software. Cells were analyzed on a BD FACSAria III flow cytometer (BD Biosciences) using Flowjo VX software.

Freshly isolated lymphocytes of blood, spleen, tonsil or cultured B cells were stained in round-bottom plates for 30 min at 4 °C in a total volume of 100 µl. All fluorochrome-conjugated mouse anti-human mAbs and quantity used for flow cytometry are shown in Supplementary Table 7. Mouse anti-human PE-Cy7-anti-IgG (clone G18-145, BD Biosciences) and APC-anti-IgA (clone IS11-8E10, Miltenyi Biotec) were used for IgG and IgA surface staining. For intracellular T-bet staining, cells were surface stained, fixed using eBio fix/perm kit (eBioscience#00-5523), washed and stained in 1 × eBio fix/perm buffer. Cells were analysed on a BD LSR II flow cytometer (BD Biosciences) using FACSDiva software. Mouse IgG1 kappa Isotype Control, PE-Cyanine7 (eBioscience#25-4714-42) was used as isotype control for T-bet staining. All conditions were collected for the same amount of time, thus the number of events displayed is reflective of the relative cell number.