Maipswu10 96 well plates

To evaluate the frequency of antigen specific memory B cells, total PBMC were cultured at 2 × 10⁶ cells/ml and stimulated with CpG-B (ODN-10103; Coley) or CpG-C (ODN-2395; Invivogen) (5 μg/ml), pokeweed mitogen (PWM, 10 μg/ml, Sigma), and Staphylococcus aureus Cowan strain (SAC, 1:10000, Sigma) for 4 days in 48-well plates. As the detection of antigen specific plasma cells does not require stimulation, bone marrow cells were plated directly into ELISpot plates at 10 × 10⁶ cells/ml. To detect Ab-secreting cells, MAIPSWU10 96 well plates (Millipore) were coated with 10 μg/ml anti-human IgG (Fcγ) (Jackson ImmunoResearch) and previously stimulated PBMCs or unstimulated bone marrow cells were transferred to the plates in dilution series and incubated ON at 37°C, 5% CO2. The plates were then washed with PBS containing 0.05% Tween and incubated with biotinylated gp140-F (strain YU2) followed by washing and incubation with streptavidin-AP (1:1000, Mabtech). The reactions were developed using BCIP/NBT substrate (Sigma) and stopped by washing in water. Spots corresponding to Ab-secreting cells (ASC) were counted using an ImmunoSpot^R analyzer (Cellular Technology Ltd.). The results were converted to Ab secreting cells per million cultured PBMCs or plated bone marrow cells (ASC/10⁶ cells), and the frequency of antigen-specific cells was calculated from the total number of ASC.

Plasma cells were enumerated as previously described (21 (link)). In brief, to evaluate the presence of BM PCs, defined as spontaneous ASC, different gate populations were sorted into supplemented media and plated directly into ELISpot plates. To detect ASC, MAIPSWU10 96-well plates (Millipore) were coated with 10 μg/ml antihuman IgG, IgA, and IgM (Fcγ, Fcα, and Fcμ chain) (Jackson ImmunoResearch), and cells were transferred to the plates in dilution series and incubated for 18 h at 37°C, 5% CO₂. The plates were then washed with PBS containing 0.05% Tween and incubated with biotinylated antihuman IgG, IgA, or IgM (0.25 μg/ml) for 1.5 h at 37°C followed by washing and incubation with streptavidin-AP (1:1000, Mabtech). The reactions were developed using BCIP/NBT substrate (Sigma) and stopped by washing in distilled water. Spots corresponding to ASC were counted using an ImmunoSpot^® analyzer (Cellular Technology Ltd.). The results were converted to show percentage of ASC of plated events.