Bambanker media

iPSCs were obtained from the iPSC repository of the DIAN project, directed by Dr. Karch (Washington University, St. Louis, MO, USA) [49 (link)]. Donor line information is detailed in Table 1. iPSC lines were thawed into Matrigel-coated 6-well plates and maintained by feeding every other day with Stemflex supplemented media (Thermo Fisher Scientific, Waltham, MA, US) at 37 °C, 5% CO₂. When 70–80% confluence was reached, iPSCs were passaged using ReLeSR dissociation reagent (#05872, STEMCELL Technologies, Vancouver, Canada). Hematopoietic stem cells were differentiated from iPSCs using the STEMdiff Hematopoietic kit (#05310 STEMCELL Technologies, Vancouver, Canada,) according to a previously published protocol [41 (link)]. When cultured for a minimum of 10 days, HPCs were collected and, if viability was >75%, frozen in Bambanker media (Wako Chemicals, Osakan, Japan).

The guinea pig used in this study, designated as 1567, was immunized in a previous study (Feng et al., 2016 (link)). Briefly, along with another five guinea pigs in the same group, animal 1567 was immunized four times at weeks 0, 4, 12, and 24 with HIV-1 Env trimer BG505 SOSIP formulated in ISOMATRIX adjuvant. Blood samples were harvested 2 weeks after each immunization with the terminal bleed on week 46 to prepare for PBMC and sera for downstream analysis (Feng et al., 2016 (link); Figure 1A,B). Four days prior to the termination, an inoculation of 40 μg of Env trimer BG505 SOSIP in the absence of adjuvant was administered by intraperitoneal injection (IP) route. The PBMCs from whole blood were further purified by density gradient centrifugation with Ficoll-Paque PLUS (GE Healthcare). After washing by PBS, cells were resuspended and frozen gradually in Bambanker media (Wako Chemicals) at -80°C followed by storage in liquid nitrogen prior to the staining and sorting experiment. The animal study was carried out at Covance with the protocol approved by the Covance Institutional Animal Care and Use Committee (IACUC, protocol #0138-14).