Sac pansorbin cells

2×10⁵ cells/well of isolated PBMCs were seeded into 96-well, cell culture treated plates (Greiner-One Bio, UK) and stimulated with 100 µl/well of a mixture of 1∶5000 Staphylococcus aureus Cowan 1 strain (SAC Pansorbin cells, Merck-Millipore, UK), 1.7 µg/ml CpG (BioScience Ltd, UK) and 83.33 ng/ml pokeweed mitogen (Sigma-Aldrich, UK). This combination induces polyclonal stimulation of B-cells and maximal proliferation of memory B-cells allowing differentiation of small antigen-specific populations into IgG-ASCs that may be detected by ELISpot [14] (link), [15] (link). Plates were incubated at 37°C, 5% CO₂ and 95% humidity for 5–6 days. Harvested cells were washed in phosphate buffered saline with ethylenediaminetetraacetic acid (EDTA) di-sodium and 0.5% newborn bovine serum (Sigma-Aldrich, UK) and re-suspended in R10 medium to a concentration of 2×10⁶ cells/ml.

For cell culture, PBMCs were thawed at 37°C in a water bath and added to warmed 15 mL cell recovery medium with 10 μL benzonase. Cells were washed twice and resuspended at 2 × 10⁶ in RPMI 1640 containing 5 mL Penicillin/Streptomycin and 5 mL l-Glutamate and 10% fetal bovine serum (FBS). Cells were stimulated with 1/5000 dilution Staphylococcus aureus Cowan 1 strain (SAC Pansorbin cells, Merck-Millipore, UK), 83 ng/mL Pokeweed mitogen (Sigma-Aldrich, UK) and 2.5 μg/mL CpG oligonucleotide at 37°C/5%CO₂/95% humidity for 5 days. Cells were then washed 4 times and resuspended in medium with10% FBS. ELISPOT was performed as described above.