After the enrichment with Pfs25 and decoy tetramers, human cells were stained with CD3- (UCHT1), CD14- (M5E2), CD56- (HCD56) Alexa Fluor 700–, CD19 APC-CY7– (HIB19), and CD20 PE-CY7–conjugated (2H7) Abs purchased from BioLegend.
A gating strategy was performed first for doublet discrimination, then singlet cells were selected for exclusion of non–B cells using CD3, CD14, and CD56 markers. Lymphocytes were gated for CD19+CD20+. Pfs25-specific B cells were gated using PE and excluding non-Pfs25 B cells in CF594, the fluorochrome used in the decoy BSA tetramer. Analysis was performed in FACSAria II instrument (BD Biosciences) with blue, red, and violet lasers. Pfs25-specific B cells were analyzed according to the fluorescence staining profile previously described and sorted directly into a 96-well plate using FACS with a nozzle of 100 μM. Plates were then centrifuged at 1278g for 30 seconds and placed into a –80°C freezer.
A gating strategy was performed first for doublet discrimination, then singlet cells were selected for exclusion of non–B cells using CD3, CD14, and CD56 markers. Lymphocytes were gated for CD19+CD20+. Pfs25-specific B cells were gated using PE and excluding non-Pfs25 B cells in CF594, the fluorochrome used in the decoy BSA tetramer. Analysis was performed in FACSAria II instrument (BD Biosciences) with blue, red, and violet lasers. Pfs25-specific B cells were analyzed according to the fluorescence staining profile previously described and sorted directly into a 96-well plate using FACS with a nozzle of 100 μM. Plates were then centrifuged at 1278g for 30 seconds and placed into a –80°C freezer.