Microamp endura optical 96 well clear reaction plates

PBMCs were combined from three pneumococcal vaccine recipients aged 25 to 42 (two Pneumovax and one PCV13 recipient) to increase the probability of isolating PPS3-specific memory B cells. PPS3-memory B cells were defined as CD19⁺CD27⁺IgM⁻IgG⁺PPS3⁺. PBMCs were stained with PPS3-PE and anti-human fluorescently conjugated CD19-PE-Cy7, CD27-APC, IgM-FITC, IgG-V421, CD3-V500, CD4-V500, CD8-V500, and CD14-V500 (BD). Live/dead (LD) cells were identified with the Zombie aqua fixable viability kit (Biolegend). CD3-, CD4-, CD8-, and CD14-positive cells were excluded. The gating strategy is shown in Fig. S8 in the supplemental material. Single cells were sorted on a BD FACSAria II instrument into 96-well PCR plates (MicroAmp Endura Optical 96-well clear reaction plates; Life Technologies) into lysis buffer as described (50 (link)).

PBMCs were combined from three pneumococcal vaccine recipients aged 25 to 42 (two Pneumovax and one PCV13 recipient) to increase the probability of isolating PPS3-specific memory B cells. PPS3-memory B cells were defined as CD19⁺CD27⁺IgM⁻IgG⁺PPS3⁺. PBMCs were stained with PPS3-PE and anti-human fluorescently conjugated CD19-PE-Cy7, CD27-APC, IgM-FITC, IgG-V421, CD3-V500, CD4-V500, CD8-V500, and CD14-V500 (BD). Live/dead (LD) cells were identified with the Zombie aqua fixable viability kit (Biolegend). CD3-, CD4-, CD8-, and CD14-positive cells were excluded. The gating strategy is shown in Fig. S8 in the supplemental material. Single cells were sorted on a BD FACSAria II instrument into 96-well PCR plates (MicroAmp Endura Optical 96-well clear reaction plates; Life Technologies) into lysis buffer as described (50 (link)).