GSM7385329: SpikeIn_5_DGE; Homo sapiens; RNA-Seq
Whole human blood was obtained from Research Blood Components, LLC (Watertown, MA), and PBMC were isolated by means of density gradient centrifugation and cryopreserved. Upon use, PBMC were thawed into Aim-V medium (ThermoFisher). B cells were isolated using an EasySep Human Pan-B Cell Enrichment kit (Stemcell Technologies, Vancouver, Canada). Biotinylated pneumococcal serotype 3 polysaccharide with a 5% biotin load (ST3, from Pfizer EPBD) was coupled with high concentration streptavidin-phycoerythrin (SA-PE) and streptavidin-allophycocyanin (SA-APC, both from BioLegend) in separate reactions. Biotinylated ST3 and the fluorophore-conjugated streptavidin reagents were prepared with PBS at 4X working solutions of 20 µg/mL and 200 µg/mL, respectively. Equal volumes of ST3 and SA-PE; and, separately, equal volumes of ST3 and SA-APC working solutions were combined, thoroughly mixed and incubated at 4 ºC protected from light for a minimum incubation period of 60 minutes with thorough mixing at 30 minutes and directly prior to use. Coupled ST3-PE and ST3-APC solutions were maintained at 4 ºC, protected from light until use. Seq-Well