CD19+ B cells were isolated (enriched) from buffy coats by MACS using CD19 magnetic microbeads (Miltenyi Biotec GmbH, cat. no. 130-050-301) and were collected in prewarmed B-cell medium. B cells were cocultured with K562 cells or K562_h4-1BB cells at a 1:1 ratio (2.5×105 cells/mL for both cell types) in the presence of DuoBody-CD40×4-1BB at 37°C, 5% CO2 for 2 days. Expression of activation markers CD69 and CD86 was evaluated by flow cytometry.
Cd19 magnetic microbeads
Manufactured by Miltenyi Biotec
CD19 magnetic microbeads are uniform superparamagnetic particles coated with antibodies specific for the human CD19 antigen. They are designed for the positive selection of CD19+ B cells from various sample types.
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Lab products found in correlation
Facsaria 3, by BD (2 mentions)
Cd43 magnetic microbeads, by Miltenyi Biotec (1 mentions)
Ls column, by Miltenyi Biotec (1 mentions)
Ms column, by Miltenyi Biotec (1 mentions)
Cd38 pe, by Beckman Coulter (1 mentions)
Cd3 apc cy7, by BioLegend (1 mentions)
Cd27 bv650, by BioLegend (1 mentions)
Cd19 pe cy7, by BD (1 mentions)
Flow cytometry staining buffer, by BD (1 mentions)
Zombie red fixable viability kit, by BioLegend (1 mentions)
Near ir dead cell stain kit, by Thermo Fisher Scientific (1 mentions)
7 aminoactinomycin d 7 aad, by Thermo Fisher Scientific (1 mentions)
Cd141, by Miltenyi Biotec (1 mentions)
4 protocols using cd19 magnetic microbeads
1
B-cell Activation by CD40×4-1BB
2
Isolation and Purification of Immune Cells
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Single cell suspensions from the bone marrows or spleens were obtained by standard techniques. Rag2-deficient pro-B cells (from the bone marrow of Rag2-/- mice) and WT fetal liver B cells (at day 14 post-coitum) were positively sorted by using B220- and CD19-magnetic microbeads and MS columns (Miltenyi). Splenic B cells were negatively sorted by using CD43-magnetic microbeads and LS columns (Miltenyi). Splenic CD4+ cells were sorted as B220-IgM-CD4+ population. ESCs cells were serum-grown in the presence of LIF (106 units/ml) throughout: first on mitomycin-treated feeder cells for 2 days, trypsinized and amplified for additional 2 days without feeders. After trypsinization, the cells were plated on gelatinized dishes for 2 hours, and the ESC-enriched supernatant carefully pipetted off and plated again for additional 2 hours in order to get rid of contaminating feeders. Sperm was collected from the cauda epididymis of adult males by the “swim-up” method [72 (link)].
3
Sorting Memory B Cells and Plasmablasts
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The CD19+ cell fraction was enriched from PBMCs by positive selection with CD19 magnetic microbeads (Miltenyi Biotech) and subsequently stained on ice for 20 min with the following fluorochrome-labeled mouse monoclonal antibodies: CD3-APC/Cy7 (dilution 1:40, clone HIT3a, catalog no. 300317, BioLegend), CD27-Bv650 (dilution 1:50, clone O323, catalog no. 302827, BioLegend), CD19-PE-Cy7 (dilution 1:50, clone SJ25C1, catalog no. 341113, BD Biosciences), HLA-DR-BD Horizon V500 (dilution 1:100, clone G46-6, catalog no. 561224, BD Biosciences) and CD38-PE (dilution 1:100, clone T16, catalog no. IM1832U, Beckman Coulter). Cells were sorted to over 98% purity on a FACSAria III (BD) using the following gating strategy: circulating memory B cells were sorted as CD3–CD19+CD27+CD38−/+ cells, whereas circulating plasmablasts were sorted as CD3–CD19+CD27hiCD38hi cells. FACS-sorted cells were collected in 6 μl FCS in Eppendorf tubes that were pre-coated overnight with 2% BSA.
4
Multiparameter PBMC Profiling by FACS
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For in vitro DC-T-cell coculture and NanoString nCounter gene expression analysis, PBMCs were directly used for FACS. CD19+ cells were depleted before sorting using CD19 magnetic MicroBeads (Miltenyi Biotec) according to the manufacturer’s protocol. Staining was performed at 4 °C for 45 min in flow cytometry staining buffer (BD Biosciences). Antibodies specific for the following proteins were used: from BioLegend, CD1c (PE-Cy7, clone L161), CD3 (BV510, clone OKT3), CD4 (FITC, clone OKT4), CD8 (Percp-cy5.5, clone SK1), CD11c (PE/AF700, clone Bu15/3.9), CD14 (ef450, clone M5E2), CD19 (BV510, clone HIB19), CD25 (PE, clone BC96), CD45RA (AF700, clone HI100), CD141 (BV421, clone M80), and HLA-DR (BV605, clone L243); from BD Biosciences, HLA-DR (APC-Cy7, clone G46-6); and from Miltenyi Biotec, CD141 (APC, clone REA674). A Near-IR Dead Cell Stain Kit (Invitrogen), a Zombie Red Fixable Viability Kit (BioLegend) or 7-aminoactinomycin D (7-AAD, eBioscience) was used to exclude dead cells. To prevent clumping of dead cells, DNase I (0.1 mg/ml) was added before sorting. Cell sorting was performed on a BD FACSAria III (BD Biosciences).
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