Disposable micropestles

For flow cytometry and cell sorting, disposable micropestles (Axygen) were used to mechanically disassociate LNs into cell suspensions. Cells from each tissue were resuspended in PBE 1X (PBS supplemented with 0.5% BSA + 1 mM EDTA) and incubated for 30 min on ice with fluorescently-labeled antibodies described in the Key Resources Table. Cells were filtered and washed with PBE again before analysis on BD FACS LSR II or FACS Symphony cytometers and sorting on FACS ARIA II cytometers. Analyses were performed using FlowJo v. 10 software.

Popliteal lymph nodes were harvested, incubated 30 min at 37 °C in RPMI, 2% FBS, 20 mM HEPES, 400 U/ml type 4 collagenase (Worthington Biochemical Corporation), disrupted using disposable micropestles (Axygen) and filtered through a 70 μm cell strainer. Single-cell suspensions were washed with PBS, 0.5% BSA, 2mM EDTA (PBE), incubated at RT for 5 minutes with 1 μg/ml of anti-CD16/32 (2.4G2, BioXCell) and then stained for cell surface markers at 4 °C for 15 min in PBE using the reagents listed in Supplementary Table 3. Cells were washed with PBS and stained with Zombie fixable viability dyes (Biolegend) at RT for 15 min and then fixed with Cytofix (BD Biosciences) before acquisition. In all in vivo experiments involving detection of Biotin-LPETG SrtA substrate, anti-biotin PE antibody (Miltenyi Biotec) was exclusively used due to its lower background compared to Streptavidin conjugates. To eliminate unspecific signal derived from PE binding by a fraction of the B cell population and thus reduce background, PE-Cy7 isotype control⁺ cells were excluded from analysis. In all in vivo experiments involving detection of CD40L, biotinylated anti-CD40L antibody (eBioscience) followed by anti-biotin PE antibody (Miltenyi Biotec) was used. Samples were acquired on Fortessa or LSR-II flow cytometers (BD Biosciences) and data were analyzed using FlowJo v.10.0.8 software.