Circulating CXCL13 in vivo and CXCL13 expression and presentation by ECs in vitro. (A) WT C57BL/6 mice were immunized with rhfF8 by intraperitoneal injection with IFA. Blood samples from immunized and paired unimmunized controls were collected 7 days later and plasma CXCL13 was measured by ELISA. (B-C) hDMVECs were cultures in the absence (control) or presence of 100 ng/mL recombinant human inflammatory cytokines (TNF-α, IFN-γ, IL1-β) or 10 ng/mL LPS for 24 hours. Alternatively, untreated control hDMVECs were incubated with 100 ng/mL recombinant human CXCL13 for 1 hour. Cells were then rinsed, fixed, and stained (without permeabilization) with anti-CXCL13 antibody. (B) Representative images (for select conditions) with anti-CXCL13 antibody staining shown in green. Scale bar, 10 μm. (C) For each condition, mean fluorescence intensity (MFI) of CXCL13 staining (representing endogenous expression and cell-surface presentation of CXCL13 or capture/presentation of exogenous CXCL13) was quantified per field of view for a total of 10 fields of view for each of 2 separate experiments and averaged.