Red blood cell lysis buffer hybri max

Mice were sacrificed by CO₂ asphyxiation. A mid‐sagittal incision was made on the abdominal cavity. The spleen was removed and passed through a 100‐μm cell strainer (Corning Incorporated, Corning, NY, USA). The single‐cell suspension was washed in Roswell Park Memorial Institute Medium (RPMI; Life Technologies) + 100 μg mL⁻¹ penicillin and streptomycin (ps; Gibco^®, Life Technologies) (RPMI/ps) and incubated for 7 min in Red Blood Cell Lysis Buffer Hybri‐Max™ (Sigma‐Aldrich^®). Cells were washed in RPMI/ps, pelleted by centrifugation and counted on the Countess II FL (Life Technologies), as per manufacturer's protocol. The liver was perfused with 1× phosphate‐buffered saline (PBS). The excised liver was collected in 1% (v/v) foetal bovine serum (FBS) in PBS and mechanically passed through a 200‐μm square metal mesh. Hepatocytes were separated from lymphocytes and removed using a 33% Percoll™ (GE Healthcare) gradient according to manufacturer's instructions. Red Blood Cell Lysis Buffer Hybri‐Max™ was added to each pellet and incubated for 5 min at room temperature, prior to washing in 1% (v/v) FBS in PBS. Cells were pelleted by centrifugation and counted using the Countess II FL, as per manufacturer's protocol.

Donor C57BL6/J mice were immunized for EAE as described above but without the pertussis toxin. Spleens and draining lymph nodes were harvested at 12 d.p.i, and single-cell suspensions were made by passage through a 70-μm cell strainer (BD Biosciences, Franklin Lakes, NJ). Red blood cells were lysed using Red Blood Cell Lysis Buffer Hybri-Max (Sigma-Aldrich, St. Louis, MO). Splenocytes were washed and re-suspended in culture medium containing Dulbecco’s modified Eagle medium (DMEM), 100 U/mL penicillin, 100 μg/mL streptomycin, 10 mM HEPES, 2 mM l-glutamine, 50 μM 2-mercaptoethanol, non-essential amino acids and 10% fetal calf serum (all from Life Technologies, Carlsbad, CA, USA). Donor cells were cultured in tissue culture flasks at 1 × 10⁷ cells/mL with IL-12p70 (20 ng/mL; Peprotech, Rocky Hill, NJ, USA), XMG1.2 (10 μg/mL; BioXCell, USA), MOG_35-55 (50 μg/mL), and either vehicle or clozapine (20 μM) at 37 °C and 5% CO₂. After 96 h of stimulation, non-adherent donor cells were harvested and injected into recipient naïve C57BL/6J mice (2 × 10⁷ cells/mouse) followed by pertussis toxin (200 ng/mouse) on days 0 and 2. Mice were monitored daily for disease as described above.