Figure 2
ELISpot procedure to analyze the number of IgM-secreting cells. Porcine PBMC were incubated in cell culture medium alone or with (i) rIdeSsuis_homologue (rIdeSsuis_h), (ii) rIdeSsuis_homologue_C195S (rIdeSsuis_h_C195S), (iii) rIdeSsuis_C_domain, or (iv) rMRP for 45 min before protein removal by washing. Treatment with these recombinant proteins was performed before (A) or after (B) 3 d of incubation in stimulation medium (supplemented with IL-2 and R848). After protein treatment and subsequent stimulation, the culture supernatant was collected for IgM ELISA, and the cells that were not used for ELISpot analysis were analysed via flow cytometry. The cells were washed and readjusted before being seeded in the ELISpot plate at 1 × 104 cells/well. The wells were coated with an anti-IgM antibody to capture secreted IgM. After 24 h, the cells were removed, and the captured IgM was detected with a second, biotinylated anti-IgM antibody. Streptavidin transforms the added substrate to a purple color, which forms visible spots exactly at the place where an IgM-secreting cell has been. This figure was created with Biorender.