Pansorbin cells

Blood lymphocytes were purified from Buffy coats (Blood Bank, Karolinska University Hospital, Stockholm, Sweden) by Ficoll-Paque (Lymphoprep, Axis-shield PoC AS, Oslo, Norway) density gradient centrifugation and B cells were affinity-purified using CD19 micro beads (MACS MicroBeads, Miltenyi Biotec, Bergisch Gladbach, Germany) resulting in >95% pure B-cell populations. B cells were infected with B95-8 virus containing supernatant for 1.5 h at 37 °C at a concentration of 2 × 10⁶/ml (3 × 10⁵ infectious units) and then diluted at the desired concentration without removal of the virus. For mitogen stimulation, the cultures were supplemented with heat-killed and formalin-fixed Staphylococcus aureus (1/20000 pansorbin cells, Calbiochem, Merck KGaA, Darmstadt, Germany) and 20 U/ml recombinant IL-2 (Peprotech, Rocky Hill, NJ, USA). Both B-cell cultures were maintained in RPMI-1640, supplemented with 20% fetal bovine serum and 10 μg/ml Ciprofloxacin. For cell proliferation, 10⁵ cells were cultured in 200 μl medium in 96 well plates, while for protein analysis, 10⁶ cells were cultured in 2 ml medium in 24 well plates.

All IP experiments were performed using IPN tissue obtained from prepubescent mice and rats (17–19 days of age) following decapitation. The protocols for solubilizing, isolating and labelling the receptors with [³H]-epibatidine were as published previously (David et al., 2010 (link); Scholze et al., 2012 (link)). Here, we used standardized Pansorbin cells (catalog number 507861; Calbiochem, Merck-Millipore, Darmstadt, Germany) for IP and the bicinchoninic acid protein assay reagent kit (Thermo Scientific Pierce, Rockford, IL, USA) for protein quantification.