F4 80 apcef780

One million cells were stained in PBS+1% FBS. Cocktails of antibodies against the following surface proteins were used: B220 PerCP-Cy5.5 (RA3-6B2), B220 FITC (RA3-6B2), GL7 BV421 (GL7), IgD BV786 (11-26c.2a), Ly6G PE (1A8), CD11b PerCP-Cy5.5 (M1/70), CD11b PE-Cy7 (M1/70), CD4 APC (RM4-5), CxCR5 BV421 (2G8), CD11c BV421 (HL3) (All BD) CD38 PE-Cy7 (90), F4/80 PE-Cy7 (BM8), F4/80 APC-EF780 (eBioscience), CD11c APC-Cy7 (N418), and PD-1 BV605 (29F.1A12) (Biolegend). A live/dead marker was used to exclude dead cells in the GC B and TFH cell panels (Fixable Viability Dye eFluor™ 780, eBioscience). AF647-labeled ovalbumin (OVA-AF647) was from Invitrogen. Antigen-specific germinal center B cells were measured by including CTH522 coupled to AF488 as probe (conjugated by Life technologies at a coupling ratio of 3 moles dye/mole). Cells were analyzed on a BD Fortessa or FACSCanto flow cytometer.

Mice were anesthetized, livers perfused with sterile PBS, and non‐parenchymal liver cells isolated as previously described (with slight modifications).24 Briefly, after successful perfusion, whole livers were excised and minced, incubated with 0.2 mg/mL collagenase IV buffer at 37°C for 30 minutes with shaking, double‐passed through 70 μm nylon mesh and centrifuged twice for 3 minutes at 40 g to pellet hepatocytes. Supernatants containing non‐parenchymal cells were purified in the Percoll gradient (Sigma‐Aldrich). Isolated cells were counted in hemocytometer (Bürker‐Türk chamber) and labelled with fluorescent‐conjugated antibodies: CD45‐APC (eBioscience), CD3‐APCeF780 (eBioscience), B220‐PECy7 (eBioscience), NK1.1‐PE (eBioscience), CD11b‐PECy7 (eBioscience), F4/80‐APCeF780 (eBioscience), Ly6G‐PE (BD Pharmingen) and CD95‐AF488 (eBioscience). CD16/CD32 (eBioscience) was used for blocking of Fc receptors, while dead cells were excluded based on 7‐amino‐actinomycin D (7‐AAD; BioLegend) binding. Upon labelling, cells were acquired and analysed using the Attune instrument (Thermo Fisher Scientific) and FlowJo software (FlowJo). The gating strategy is shown in Supporting Information (Figure S1).