Transfer of HEL-specific B Cells

Freshly isolated bulk splenocytes from SW_HEL mice containing 10⁴ HEL⁺ B cells were transferred into C57BL/6 mice by intravenous injection, as previously described [50] (link). Sheep red blood cells (SRBC) were obtained from Miguel Fevereiro, Laboratório Nacional de Investigação Veterinária, Lisbon. Recombinant HEL was covalently conjugated to SRBC with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (Sigma-Aldrich) as described [25] (link). Conjugation was confirmed by FACS by staining mock or HEL-conjugated SRBC with the HyHEL10 (an anti-HEL IgG₁[21] (link)) followed by an anti-IgG₁ APC (BD Biosciences). HEL-A647 was used to identify transferred HEL⁺ B cells.