Human memory b cell isolation kit

The cryopreserved PBMCs from Subject 009 were thawed and cultured in RPMI 1640 with 15% Fetal Bovine Serum (FBS), 1% L-glutamine and 1% Penicillin-Streptomycin (P/S) at 37°C with 5% CO2 overnight. The B cells from overnighted culture PBMCs were first enriched using MACS Human B Cell Isolation Kit II (Milteny Biotec, San Diego, CA). Then, CD27+ memory B cells were isolated using human memory B cell isolation kit (Milteny Biotec). For EBV immortalization of B cells, the memory B cells isolated from Subject 009 were seeded at 5 cells/well in 96-well U-bottom microplates in 200µl of complete RPMI medium containing 2.5µg/ml CpG ODN2006, in the presence of EBV (30% supernatant of B95-8 cells) and irradiated allogeneic mononuclear cells (50,000 per well) ^{48 (link)}. After 12 days of culture at 37°C with 5% CO2, 50µl of culture supernatants were harvested and screened for gp120 binding by ELISA. The EBV transformed cells from the gp120 binding positive wells were picked and expanded for clonal culture. The antibody from positive wells were further determined by ELISA with anti-Lamda and anti-Kappa secondary antibody, separately. The cells from gp120-specific clone #64 were harvested and preserved in RNALater (Qiagen, Redwood City, CA) for RNA extraction and Ig gene cloning.

The cryopreserved PBMCs from Subject 009 were thawed and cultured in RPMI 1640 with 15% Fetal Bovine Serum (FBS), 1% L-glutamine and 1% Penicillin-Streptomycin (P/S) at 37 °C with 5% CO₂ overnight. The B cells from overnight cultured PBMCs were first enriched using MACS Human B Cell Isolation Kit II (Miltenyi Biotec, San Diego, CA). Then, CD27+ memory B cells were isolated using the Human Memory B Cell Isolation Kit (Miltenyi Biotec). For EBV immortalization of B cells, the memory B cells isolated from Subject 009 were seeded at 5 cells per well in 96-well U-bottom microplates in 200 µl of complete RPMI medium containing 2.5 µg/ml CpG ODN2006, in the presence of EBV (30% supernatant of B95-8 cells) and irradiated allogeneic mononuclear cells (50,000 per well)^{53 (link)}. After 12 days of culture at 37 °C with 5% CO2, 50 µl of culture supernatants were harvested and screened for gp120 binding by ELISA. The EBV-transformed cells from the gp120 binding positive wells were picked and expanded for clonal culture. The antibody from positive wells was further determined by ELISA with anti-lamda and anti-kappa secondary antibodies separately. The cells from gp120-specific clone #64 were harvested and preserved in RNAlater (Qiagen, Redwood City, CA) for RNA extraction and Ig gene cloning.

Total B cells were isolated from PBMCs using the Human Memory B Cell Isolation Kit (Miltenyi Biotec) with an LD column according to the manufacturer’s instructions. B cells were then coemulsified with oligo d(T)25 magnetic beads (New England Biolabs) and lysis buffer (100 mM Tris pH 7.5, 500 mM LiCl, 10 mM EDTA, 1% lithium dodecyl sulfate, and 5 mM dithiothreitol) using a custom-designed flow focusing device as described (McDaniel et al., 2016 (link)). The magnetic beads were washed, resuspended in One step fast qRT-PCR solution (VWR) supplemented with an overlap extension VH and VL primer set as described (McDaniel et al., 2016 (link)), emulsified, and subjected to overlap-extension RT-PCR under the following conditions: 30 min at 55°C followed by 2 min at 94°C; 4 cycles of 94°C for 30 s, 50°C for 30 s, 72°C for 2 min; 4 cycles of 94°C for 30 s, 55°C for 30 s, 72°C for 2 min; 32 cycles of 94°C for 30 s, 60°C for 30 s, 72°C for 2 min; 72°C for 7 min; hold at 4°C. VH/VL amplicons were then extracted from the emulsions, amplified using nested PCR, and sequenced by 2x300 Illumina MiSeq.