Human peripheral blood was obtained from healthy adult donors and provided by the General Hospital of Southern Theatre Command. All participants provided written informed consent to participate in the study. Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood by Ficoll (TBD Science) density-gradient centrifugation. Naïve B cells were isolated from PBMCs using an EasySep Human Naïve B Cell Isolation Kit (17254, STEMCELL Technologies) according to the manufacturer’s protocol. These experiments were performed according to the guidelines of the Research Ethics Committee of Guangdong Provincial People’s Hospital (No.GDREC2020078H(R2)).
Easysep human na ve b cell isolation kit
Manufactured by STEMCELL
The EasySep Human Naïve B Cell Isolation Kit is a magnetic cell separation system designed to isolate naïve B cells from human peripheral blood mononuclear cells (PBMCs). The kit uses immunomagnetic particles to negatively select for naïve B cells by targeting and removing non-naïve B cells and other unwanted cell types.
Automatically generated - may contain errors
Lab products found in correlation
Ficoll, by TBD Science (1 mentions)
Penicillin streptomycin glutamine (psg), by Thermo Fisher Scientific (1 mentions)
Anti cd40 antibody, by BioXCell (1 mentions)
Easysep human pan b cell enrichment kit, by STEMCELL (1 mentions)
Interleukin 4 (il 4), by Immunotools (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Hepes, by Thermo Fisher Scientific (1 mentions)
β mercaptoethanol, by Merck Group (1 mentions)
3 protocols using easysep human na ve b cell isolation kit
1
Isolation of Naïve B Cells from Human Peripheral Blood
2
B Cell Activation and Differentiation
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B lymphocytes were purified from the PBMC fraction using negative isolation EasySep™ Human Naïve B Cell Isolation Kit or EasySep™ Human Pan-B Cell Enrichment Kit (both Stemcell Technologies). B cells were cultured in RPMI supplemented with 10% FCS, 1%PSG (both Thermo Scientific), 1 µg/ml anti-IgM (Clone DA4-4, MyBiosource), 20 ng/ml IL-4 (Immunotools) and 1 µg/ml anti-CD40 antibody (Clone G28.5 BioXcell) for 7 days. When indicated, 1 µM retinoic acid, retinal, RA-click or RE-click was added once at day 0.
3
Isolation and Characterization of Primary Naïve Human B Cells
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Primary naive B cells were obtained from fresh buffy coats provided by the Institute for Transfusion Medicine and Gene Therapy, ITG Freiburg. Peripheral blood mononuclear cells (PBMCs) were separated by Ficoll gradient centrifugation of whole blood and negatively selected using EasySep Human Naïve B Cell Isolation kit (StemCell). Prior to the experiments, primary naïve B cells were controlled for purity and rested overnight in complete RPMI 1640 supplemented with 5% FCS, 10 units/ml penicillin/streptomycin (Gibco), 20 mM HEPES (Gibco), and 50 mM β-mercaptoethanol (Sigma) at 37°C with 5% CO2. Experiments with primary human B cells were repeated at least three times. This study was approved by the Institutional Review Board of the University Freiburg (Ethical votes No 507/16 and 336/16).
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