Alexa fluor 647 conjugated goat anti human igm

Cryopreserved PfSPZ (Sanaria^®) were thawed and stained with different concentrations of Tanzanian sera or monoclonal antibodies in 3.3× SYBR Green I (ThermoFisher Scientific) for 30 min at 4°C. The PfSPZ were washed twice by centrifugation at 3220 × g for 5 min. Human serum antibody binding was detected using 2.5 μg ml⁻¹ Alexa Fluor 647-conjugated goat anti-human IgG (Jackson ImmunoResearch, 109-606-170) or Alexa Fluor 647-conjugated goat anti-human IgM (Jackson ImmunoResearch, 109-606-129). Mouse serum antibody binding was detected using 1 μg ml⁻¹ PE-Cy7-conjugated goat anti-mouse IgG (Biolegend, 405315) or PE-Cy7-conjugated rat anti-mouse IgM (BD Biosciences, 552867). FACS Diva (version 6.2) was used for acquisition of samples and Flow-Jo (version 10.1) was used for FACS analysis. The PfSPZ were gated based on high fluorescence in the FITC channel. Median fluorescence intensity (MFI) of the PfSPZ in the Alexa Fluor 647 or PE-Cy7 channel was calculated to quantify IgG or IgM binding. The concentration of antibody needed to achieve MFI 10,000 (Conc₁₀₀₀₀) was calculated by interpolation of binding curves fitted to a sigmoidal curve model (Graphpad Prism 7) as a measure of affinity. The gating strategy can be found in Supplementary Figure 1.