For induction of GC reactions, each mouse was intravenously injected with 200 μl of the SRBC suspension containing 2 × 108 SRBCs, and spleens were analyzed at day 10. Alternatively, Plasmodium berghei parasites engineered to express Plasmodium falciparum CSP (PfCSP) in place of the endogenous P. berghei CSP molecule (Pb-PfSPZ) (51 (link)) were used. Parasites were maintained by serial passage through Anopheles stephensi mosquitoes. Mice were immunized intravenously with 5 × 104 irradiated (15 kRad) Pb-PfSPZ dissected by hand from the salivary glands of A. stephensi mosquitoes as described previously.
For the detection of PfCSP-specific cells, a nine-times repeat of asparagine-alanine-asparagine-proline (NANP)9 peptide was sourced from Biomatik (Ontario, Canada) and was biotinylated with the Sulfo-NHS-LC Biotinylation Kit (Thermo Fisher Scientific) at a ratio of 1:1 according to the manufacturer’s instructions. Biotinylated antigens were incubated with premium-grade streptavidin-phycoerythrin and streptavidin-allophycocyanine (Molecular Probes) at a molar ratio of 4:1 and added four times with 15-min incubation at room temperature.
For the detection of PfCSP-specific cells, a nine-times repeat of asparagine-alanine-asparagine-proline (NANP)9 peptide was sourced from Biomatik (Ontario, Canada) and was biotinylated with the Sulfo-NHS-LC Biotinylation Kit (Thermo Fisher Scientific) at a ratio of 1:1 according to the manufacturer’s instructions. Biotinylated antigens were incubated with premium-grade streptavidin-phycoerythrin and streptavidin-allophycocyanine (Molecular Probes) at a molar ratio of 4:1 and added four times with 15-min incubation at room temperature.
