Memory B cells were differentiated in vitro into plasmablasts and plasma cells, following the protocol described in Jourdan et al. (24 (link)). Briefly, CD27+ Memory B cells were first purified as bulk B cells by negative selection from PBMCs by magnetic isolation followed by positive selection for CD27+ cells (Stemcell Technologies, UK) following manufacture's protocol. Then seeded at1.5 × 105 cells/ml, and cultured for 4 days in the presence of CpG oligodeoxynucleotide 2006 (10 μg/ml; Invitrogen), soluble human his-rCD40L (50ng/ml; R&D Systems, UK) and anti-poly-his (5μ g/ml; R&D Systems, UK), IL-2 (20 U/ml; R&D Systems, UK), IL-10 (50 ng/ml; Miltenyi Biotec, UK) and IL-15 (10 ng/ml; Miltenyi Biotec, UK). At day 4 of culture, the cells were washed and cultured at 2.5 × 105 cells/ml with IL-2 (20 U/ml, Milteny Biotec, UK), IL-10 (50 ng/ml), IL-15 (10 ng/m, PreproTech, UK) and IL-6 (50ng/ml; Miltenyi Biotec, UK). At day 7 of culture, cells were washed and seeded with IL-6 (50 ng/ml, PreproTech, UK), IL-15 (10 ng/ml, PreproTech, UK) and IFN-α (500 U/ml; R&D Systems) for 3 days.
Cpg oligodeoxynucleotide 2006
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
CpG oligodeoxynucleotide 2006 is a synthetic DNA molecule composed of cytosine-phosphate-guanine (CpG) motifs. It is designed to activate the innate immune system by binding to Toll-like receptor 9 (TLR9).
Automatically generated - may contain errors
Lab products found in correlation
Il 10, by Thermo Fisher Scientific (4 mentions)
Il 15, by Thermo Fisher Scientific (4 mentions)
Cd27 cells, by STEMCELL (2 mentions)
Soluble human his rcd40l, by R&D Systems (2 mentions)
Anti poly his, by R&D Systems (2 mentions)
Il 10, by Miltenyi Biotec (2 mentions)
Interleukin 6 (il 6), by Miltenyi Biotec (2 mentions)
Interleukin 6 (il 6), by Thermo Fisher Scientific (2 mentions)
Ifn α, by R&D Systems (2 mentions)
Interleukin 2 (il 2), by Miltenyi Biotec (2 mentions)
Il 15, by Miltenyi Biotec (2 mentions)
Interleukin 2 (il 2), by R&D Systems (2 mentions)
Interleukin 2 (il 2), by Thermo Fisher Scientific (1 mentions)
B cell isolation kit 2, by Miltenyi Biotec (1 mentions)
Interleukin 2 il 2, by Thermo Fisher Scientific (1 mentions)
Megacd40l, by Enzo Life Sciences (1 mentions)
4 protocols using cpg oligodeoxynucleotide 2006
1
Differentiation of Memory B Cells into Antibody-Secreting Cells
2
CRISPR Editing of Activated B Cells
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PBMCs were collected from whole blood of consented donors and cryopreserved at the Fred Hutchinson Cancer Research Center. CD19+ B cells were subsequently isolated by negative selection (Pan-B cell kit, Miltenyi Biotec) and cultured in Iscove’s modified Dulbecco’s medium (IMDM) (Thermo Fisher Scientific) supplemented with 10% FBS and 55 μM beta-mercaptoethanol at 1 × 106 cell/mL to 1.5 × 106 cells/mL. B cells were activated with 100 ng/mL recombinant human MEGACD40L (Enzo Life Sciences), 1 μg/mL CpG oligodeoxynucleotide 2006 (Invitrogen), 50 ng/mL IL-2 (PeproTech), 50 ng/mL IL-10 (PeproTech), and 10 ng/mL IL-15 (PeproTech) for 2 days. Cells were then electroporated with Cas9 RNP complexes (see Supplemental Materials and Methods ).
3
Generating Plasma B Cells from PBMCs
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PBMCs were collected from whole blood of consented donors and cryopreserved at the Fred Hutchinson Cancer Research Center. B cells were isolated by negative selection as per manufacturer’s instructions (B cell isolation kit II, Miltenyi Biotec) and then cultured in IMDM supplemented with 10% FBS and 55 mM β-mercaptoethanol (aIMDM) at 1.5 × 106 cells/mL. B cells were activated with 100 ng/mL recombinant human MEGACD40L (Enzo Life Sciences), 1 μg/mL CpG oligo-deoxynucleotide 2006 (Invitrogen), 50 ng/mL interleukin-2 (IL-2) (PeproTech), 50 ng/mL IL-10 (PeproTech), and 10 ng/mL IL-15 (PeproTech) (cIMDM) for 2 days, before transduction with LV. Cells (3 × 105) were seeded in a 96-well plate, mixed with 20 μL of measles pseudotyped (HF MV) LV, and spinoculated at 400 rpm for 30 min. Transduced cells were then expanded for 5 days in aIMDM before being differentiated using a previously described three-step culture system.59 (link) Cells were normalized to a 1.5 × 106 cells/mL concentration in IL-2 (50 ng/mL), IL-6 (50 ng/mL), IL-10 (50 ng/mL), and IL-15 (10 ng/mL) containing aIMDM for 3 days to induce plasmablast phenotype differentiation. Differentiation was confirmed by flow cytometry, detailed below.
4
In Vitro Differentiation of Memory B Cells
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Memory B cells were differentiated in vitro into plasmablasts and plasma cells, following the protocol described in Jourdan et al. (24 (link)). Briefly, CD27+ Memory B cells were first purified as bulk B cells by negative selection from PBMCs by magnetic isolation followed by positive selection for CD27+ cells (Stemcell Technologies) following the manufacturer’s protocol. The cells were then seeded at 1.5 × 105 cells/ml and cultured for 4 d in the presence of CpG oligodeoxynucleotide 2006 (10 μg/ml; Invitrogen, Paisley, U.K.), soluble human his-rCD40L (50 ng/ml; R&D Systems, Abingdon, U.K.) and anti–poly-his (5 μg/ml; R&D Systems), IL-2 (20 U/ml; R&D Systems), IL-10 (50 ng/ml; Miltenyi Biotec, Surrey, U.K.), and IL-15 (10 ng/ml; Miltenyi Biotec). At day 4 of culture, the cells were washed and cultured at 2.5 × 105 cells/ml with IL-2 (20 U/ml; Miltenyi Biotec), IL-10 (50 ng/ml), IL-15 (10 ng/ml; PeproTech, London, U.K.), and IL-6 (50 ng/ml; Miltenyi Biotec). At day 7 of culture, cells were washed and seeded with IL-6 (50 ng/ml; PeproTech), IL-15 (10 ng/ml; PeproTech), and IFN-α (500 U/ml; R&D Systems) for 3 d.
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