Transwell permeable membrane inserts

Naive splenic B cells were purified using a negative selection magnetically assisted cell sorting kit (Miltenyi Biotech) according to the manufacturer’s protocol and cultured in IMDM (Gibco, Invitrogen) containing 10% heat-inactivated FBS (Wisent) and supplemented with penicillin-streptomycin (Life Technologies). Purified B cells were then incubated by themselves, with the parasite at a multiplicity of infection (MOI) of 5 or 10, or with 10 ng/mL mouse recombinant IFN-β (BioLegend) at 37°C and 5% CO₂ for the duration specified. For experiments assessing the necessity of direct contact for B cell activation, transwell permeable membrane inserts (Corning) with a pore size of 0.4 μm and a polyethylene terephthalate (PET) membrane were used to separate total B cell populations or B cell subpopulations, and only the cells in the top compartment were exposed to PKH67-labeled parasite.

Naive splenic B cells were purified using a negative selection magnetically assisted cell sorting kit (Miltenyi Biotech) according to the manufacturer’s protocol and cultured in IMDM (Gibco, Invitrogen) containing 10% heat-inactivated FBS (Wisent) and supplemented with penicillin-streptomycin (Life Technologies). Purified B cells were then incubated by themselves, with the parasite at a multiplicity of infection (MOI) of 5 or 10, or with 10 ng/mL mouse recombinant IFN-β (BioLegend) at 37°C and 5% CO₂ for the duration specified. For experiments assessing the necessity of direct contact for B cell activation, transwell permeable membrane inserts (Corning) with a pore size of 0.4 μm and a polyethylene terephthalate (PET) membrane were used to separate total B cell populations or B cell subpopulations, and only the cells in the top compartment were exposed to PKH67-labeled parasite.