Volunteers were recruited from among staff and students at the London School of Hygiene and Tropical Medicine. All subjects gave fully informed, written consent and the study was approved by the London School of Hygiene and Tropical Medicine Ethics Committee. Subjects ranged in age from 21 to 73 years and all donors confirmed that they had been vaccinated against diphtheria, tetanus and pertussis in childhood. Peripheral blood mononuclear cells (PBMC) were separated by fractionation on a Ficoll–Hypaque gradient and cryopreserved in liquid nitrogen. Frozen PBMC were thawed with pre-warmed complete medium [RPMI-1640 supplemented with 100 U/ml penicillin/streptomycin and 20 mm l -glutamine (Gibco, Lifesciences, Paisley, UK) and 10% pooled human AB serum (Sigma, Poole, UK)] (at 37°), washed several times and rested for 30 min before use.
Pooled human ab serum
Manufactured by Merck Group
Sourced in United Kingdom
Pooled human AB serum is a laboratory product that consists of serum derived from the blood of multiple human donors with the AB blood type. It provides a source of growth factors, nutrients, and other biomolecules that can be used to support the cultivation and maintenance of cell cultures in vitro.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Merck Group (1 mentions)
Sodium pyruvate, by Merck Group (1 mentions)
Cd14 microbead, by Miltenyi Biotec (1 mentions)
Cd4 t cell isolation kit, by Miltenyi Biotec (1 mentions)
Non essential amino acids (neaa), by Merck Group (1 mentions)
L glutamine, by Merck Group (1 mentions)
Human nk cell isolation kit, by Miltenyi Biotec (1 mentions)
Automacs, by Miltenyi Biotec (1 mentions)
Ficoll, by Harvard Bioscience (1 mentions)
Rat anti mouse igm microbeads, by Miltenyi Biotec (1 mentions)
Interleukin 2 (il 2), by Thermo Fisher Scientific (1 mentions)
Interleukin 4 (il 4), by Thermo Fisher Scientific (1 mentions)
Il 10, by Thermo Fisher Scientific (1 mentions)
Iscove modified dulbecco media (imdm), by Thermo Fisher Scientific (1 mentions)
Transferrin, by Merck Group (1 mentions)
3 protocols using pooled human ab serum
1
Isolation and Cryopreservation of PBMCs
2
Isolation of Immune Cell Subsets
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Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats of healthy adult donors by density gradient purification with Ficoll (Biochrom). slanMo were isolated as described previously.3 (link) Briefly, PBMCs were incubated in diluted M-DC8 hybridoma supernatant for 15 minutes at 4°C. After washing, the cells were incubated with rat anti-mouse IgM microbeads (Miltenyi) for 15 minutes at 4°C, washed again and then slanMo were purified by positive selection with an autoMACS (Miltenyi). Untouched NK cells from the remaining PBMCs were further purified using the human NK cell Isolation Kit (Miltenyi Biotech) according to the manufacturer’s instructions via autoMACS negative selection. The purity of slan+CD3− slanMo and of CD56+CD3− NK cells was ≥95%. Freshly isolated slanMo and NK cells were cultured in complete RPMI 1640 containing 2 mM L-glutamine, 1% penicillin/streptomycin, 1% non-essential amino acids (Sigma-Aldrich), 1 mM sodium pyruvate (Sigma-Aldrich), and 10% heat-inactivated pooled human AB serum (Sigma-Aldrich).
CD14 monocytes were isolated with CD14 microbeads (Miltenyi) by positive selection according to the protocol. T cell isolation was performed with the CD4 + T cell isolation kit (Miltenyi) by following the instructions and a negative selection autoMACS protocol.
CD14 monocytes were isolated with CD14 microbeads (Miltenyi) by positive selection according to the protocol. T cell isolation was performed with the CD4 + T cell isolation kit (Miltenyi) by following the instructions and a negative selection autoMACS protocol.
3
Expansion of Primary Human B Cells
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The 293T-CD40L, 293T-CD40L-sBAFF and normal HEK293T cell lines were seeded on a 12-well plate at 5×105 cells/well or 2×105cells/well, as described previously [48 (link)]. To prepare feeder cells, the cells was irradiated (95 Gy) after one day of seeding and incubated at 37°C for 12 hours to confirm growth arrest of the cells. The optimal ratio of cell line to B lymphocytes was determined by co-culturing feeder cells: B cells at ratios from 3:1 to 1:3, while the optimal density of B lymphocytes was determined from 5×104/well to 1×106/well. Every 4 days, the growing B cells were harvested and centrifuged in a Ficoll-Paque density gradient, counted, and analyzed by flow cytometry. B-cell co-cultures were performed in 12-well plate in the conditioned Iscove's Modified Dubecco's Medium (IMDM, Gibco), supplemented with 10% heat-inactivated pooled human AB serum (Sigma), 100 U/ml of penicillin, and 100 U/ml of streptomycin (Hyclone), 2 μg/ml CpG-ODN2006/2219 (Invitrogen), 0.625 μg/ml CsA (Cyclosporine A) (Sigma), 50 μg/ml transferrin (Sigma), 10 ng/mL IL-2 (Peprotech,), 10 ng/ml IL-10, and 20 ng/mL IL-4 (Peprotech).
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