Macs anti biotin magnetic beads

Mouse tissue‐derived cells were isolated from cell suspension after digestion of minced tissue with 1 mg/ml collagenase IV (GIBCO), 0.5 mg/ml Dispase (GIBCO), and 1 mg/ml DNase I (Applichem). Mouse blood cells were derived from peripheral blood after red blood cell lysis. Human immune cells were derived from density gradient separated blood polymorphonuclear cell and mononuclear cell fractions using Lympholyte‐poly solution (Cedarlane), and only autologous, sample‐matched cells were used for co‐culture. Specific cell populations were enriched by either magnetic or fluorescence‐based (FACS) sorting for cell type‐specific antibodies (see Appendix Tables S1 and S3 for detailed information). Biotinylated antibodies with MACS anti‐biotin magnetic beads and MACS columns (Miltenyi Biotec) were used for magnetic sorting. FACS sorting was done on FACS‐Aria II or FACS‐ARIA Fusion and FACS analysis on LSR Fortessa machines (all BD Biosciences).

OT2 T cells were enriched by negative depletion with biotinylated antibodies for anti-B220 clone RA3-6B2, anti-CD11b clone M1/70, anti-CD11c clone HL3, anti-CD8 clone 53-6.7, and SW_HEL B cells were enriched by negative depletion with biotinylated antibodies for anti-CD11b, anti-CD11c, anti-CD4 clone GK1.5, anti-CD43 clone S7 (all from BD Bisociences) and MACs anti-biotin magnetic beads (Miltenyi). Purity of CD4⁺ V_α2⁺ OT2 T cells was typically 70–80% and B220⁺ HEL-binding SW_HEL B cells >99% as determined by FACs analysis. 2.5 × 10⁵ CD4⁺Vα2⁺ OT2 T cells and B220⁺ HEL-binding SW_HEL B cells were adoptively transferred into age and sex matched 6–9-week-old C57BL/6 or SAP-deficient recipient mice. Recipient mice were immunised the next day by subcutaneous injection with 20 μg HEL-OVA in Sigma Adjuvant System (SAS, Sigma) in the lower flank and base of tail. For memory responses, mice that had been immunised were rested for at least 28 days and then re-challenged with 40 μg HEL-OVA in SAS injected subcutaneously in the lower flank and base of tail. HEL was conjugated to OVA_323–339 peptide (CGGISQAVHAAHAEINEAGR) (Mimotopes/Genscript) using the SMPH cross-linking agent Succinimidyl-6-([ß-maleimidopropionamido] hexanoate) (Thermo Fisher Scientific) to generate HEL-OVA^{48 (link)}.

Kaede OT2 T cells were enriched by negative depletion with biotinylated antibodies for anti-B220 clone RA3-6B2, anti-CD11b clone M1/70, anti-CD11c clone HL3, anti-CD8 clone 53–6.7, and tdtomato SW_HEL B cells (Phan et al., 2003 (link)) were enriched by negative depletion with biotinylated antibodies for anti-CD11b, anti-CD11c, anti-CD4 clone GK1.5, anti-CD43 clone S7 (all from BD Bisociences) and MACs anti-biotin magnetic beads (Miltenyi). 2.5 × 10⁵ B220⁺ HEL⁺ SWHEL tdTomato B cells and V_α2⁺ CD4⁺ Kaede OT2 cells were adoptively transferred into C57BL/6 recipients and immunised the next day with 20 μg HEL-OVA in Sigma Adjuvant System (Sigma) injected subcutaneously in the lower flank and tail base. To label SCS macrophages, we injected CD169 clone Ser-4 (UCSF Hybridoma Core) conjugated to Alexa Fluor 680 (Invitrogen), 12 hr before imaging. Mice were imaged 7 days after immunisation.