ELISPOTs to measure ex-vivo frequencies of P. chabaudi-specific ASC in BM and MBC in spleen were performed as previously described [61 (link)]. Cells were incubated for 5h on 96-wells Multiscreen-HA filter plates (Millipore) coated with the C-terminal 21kDa part of P. chabaudi merozoite surface protein 1 (MSP121), prepared as described [62 (link)], to measure the frequency of MSP121-specific ASC, or coated with affinity-purified goat anti-mouse IgG (Sigma), to measure the frequency of total IgG ASC. Spots were enumerated with an Immunospot analyzer (CTL, Germany).
For MBC ELISPOTs, spleen cells were obtained by mashing the spleens through a 70 μm filter strainer, and rbc were eliminated by treatment with lysing buffer (Sigma). Cells were polyclonally activated by incubation for 6 days in flat-bottomed 96-well plates in the presence of irradiated feeder splenocytes, LPS, and supernatant from concanavalin A-stimulated C57BL/6 spleen cells. Four-fold dilutions of splenocytes were tested in replicates of 22 wells each. Cells were then transferred to antigen-coated 96-well Multiscreen-HA filter plates (Millipore) for ASC ELISPOT performed as described above. Precursor frequencies were calculated with ELDA software [63 (link)].
For MBC ELISPOTs, spleen cells were obtained by mashing the spleens through a 70 μm filter strainer, and rbc were eliminated by treatment with lysing buffer (Sigma). Cells were polyclonally activated by incubation for 6 days in flat-bottomed 96-well plates in the presence of irradiated feeder splenocytes, LPS, and supernatant from concanavalin A-stimulated C57BL/6 spleen cells. Four-fold dilutions of splenocytes were tested in replicates of 22 wells each. Cells were then transferred to antigen-coated 96-well Multiscreen-HA filter plates (Millipore) for ASC ELISPOT performed as described above. Precursor frequencies were calculated with ELDA software [63 (link)].
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