Flow Cytometry-Based Cell Sorting for Different Circulatory B Cell Populations
Published: July 31, 2023
Abstract
Source: Tzeng, S. J. The Isolation, Differentiation, and Quantification of Human Antibody-secreting B Cells from Blood: ELISpot as a Functional Readout of Humoral Immunity. J. Vis. Exp. (2016).
In this video, we describe a protocol for the flow cytometry-based sorting of naïve B cell, memory B cell, and plasmablast plasma cell subpopulations from human peripheral blood B cells. The different B cell subpopulations can be distinguished based on fluorophore-based antibody binding to the CD19, CD27, and CD38 receptors expressed on the B cell surfaces, followed by cell sorting.
Protocol
All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board. 1. Cell Sorting for the Collection of Naïve B Cells, Memory B Cells, and Plasmablasts/Plasma Cells (PBs/PCs) Using purified human peripheral blood B cells, determine the cell number using a hemocytometer or an automatic cell counter. <…
Offenlegungen
The authors have nothing to disclose.
Materials
| 15 mL Falcon tubes | BD Falcon | 352196 | |
| Blue nylon mesh cell strainer, 40 μm | BD Falcon | 352340 | |
| Anti-human CD19-APC | Biolegend | 302212 | clone HIB19 |
| Anti-human CD27-eFluor 450 | eBioscience | 48-0279-42 | clone O323 |
| Anti-human CD38-PE-Cy7 | Biolegend | 303516 | clone HIT2 |
| Anti-human CD38-PE-Cy7 | BD Biosciences | 560677 | clone HIT2 |
| Anti-human CD45-FITC | Biolegend | 304006 | clone HI30 |
| Anti-human CD45-FITC | BD Biosciences | 555482 | clone HI30 |
| Anti-mouse/rat/human CD27- PerCP Cy5.5 | Biolegend | 124213 | clone LG.3A10 |
| Anti-human CD27-PerCP Cy5.5 | BD Biosciences | 65429 | clone L128 |
| Anti-human CD19-FITC | Miltenyi Biotec | 130-098-064 | clone LT19 |
| Anti-human CD19-FITC | GeneTex | GTX75599 | clone LT19 |
| Anti-human CD20-FITC | BD Biosciences | 555622 | clone 2H7 |
| 7-aminoactinomycin D (7-AAD) | BD Biosciences | 559925 | |
| Human IgG | Jackson ImmunoResearch | 009-000-003 |
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Diesen Artikel zitieren
Flow Cytometry-Based Cell Sorting for Different Circulatory B Cell Populations. J. Vis. Exp. (Pending Publication), e21524, doi: (2023).