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Antibody-secreting cell differentiation from human PBMC

RS Ruda de Luna Almeida Santos
LB Lin Bai
PS Pradeep K. Singh
NM Naoka Murakami
HF Hao Fan
WZ Wenhu Zhan
YZ Yingrong Zhu
XJ Xiuju Jiang
KZ Kaiming Zhang
JA Jean Pierre Assker
CN Carl F. Nathan
HL Huilin Li
JA Jamil Azzi
GL Gang Lin
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Human peripheral blood samples were obtained from five different healthy volunteers (BWH Specimen Bank). Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation using Lymphoprep (StemCell Technologies). PBMCs were cultured at 1.5 × 106 cells per mL in RPMI 1640 media (Lonza), supplemented with 10% human serum (GemCell) (10% HS-RPMI), recombinant human IL-2 10 ng per mL (or 100 U per ml, BioLegend) and R848 1 µg per mL (Cayman Chemicals) for 5 days, at 37 °C, 5% CO2. PKS21221 in DMSO, or DMSO only for control, was added to the culture media on day 5, and cells were incubated for additional 12, 24, and 48 h. Cells were then stained with surface marker antibodies and analyzed by flow cytometry. The viability and apoptosis were assessed using Annexin V Apoptosis Detection Kit (BD Biosciences).

Copyright and License information: The Author(s) ©2017
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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