OMIP-068
High‐Dimensional Characterization of Global and Antigen‐Specific B Cells in Chronic Infection
Cytometry PART A, Volume 97, Issue 10, 1037-1043 (2020)
Katherine Cascino, Mario Roederer, Thomas Liechti
Description: This 24‐color flow cytometry panel focuses on characterizing antigen‐specific B cells and precise delineation of B‐cell subsets in chronic infections and is applicable to other chronic diseases such as autoimmunity. The panel was optimized for human cryopreserved peripheral blood mononuclear cells (PBMCs). Markers were chosen to extensively distinguish B‐cell lineages (CD19, CD20, CD10, CD38, CD24, IgM, IgD, CD27, CD21, CD43, CD5). Inclusion of antigen‐specific probes was of high priority in order to assess hepatitis B virus (HBV) antigen‐specific B cells for our purposes. These probes can be readily exchanged for other pathogen‐specific probes or additional markers for the panel to be tailored to desired research questions beyond HBV. In addition, we included a comprehensive and unique set of functional markers such as chemokine receptors (CXCR3, CXCR5), co‐stimulatory molecule (CD86), Fc receptor (CD32), regulatory molecules (BTLA, CD39), and inhibitory markers associated with chronic infections (PD‐1, FcRL5, CD11c, CD22) to enable in‐depth analysis of global and antigen‐specific B cells during chronic infection.
Species: Human
Tissue: Peripheral blood
Cell Subset: B cell, B memory, Double negative b, Transitional b
Keyword: Antigen-specific b cell response, Autoimmunity, Cryopreserved pbmc, Hepatitis, Immune checkpoint
Machine: BD FACSymphony A5
Panel details
| Marker | Species | Clone | Fluorophore | Purpose | Co-expressions groups | Antigen Density |
|---|---|---|---|---|---|---|
| Dead cells | Human | Live/Dead Fix Aqua | n/a | |||
| CD11c | Human | Bu15 | Brilliant Blue 630-P2 | Inhibitory/exhaustion marker | B memory, DNB | Med |
| IgD | Human | IA6-2 | Brilliant Blue 790-P | Differentiation B cells | Transitional B | High |
| CD86 | Human | FUN-1 | Brilliant Ultraviolet 737 | Differentiation B cells | B memory | Med |
| FCRL5 | Human | 509f6 | Brilliant Blue 660-P2 | Inhibitory/exhaustion marker | B memory | Med |
| PD-1 | Human | EH12.2H7 | FITC | Inhibitory/exhaustion marker | DNB | Low |
| CD10 | Human | HI10a | PE-Cy5 | Differentiation B cells | Transitional B | Low |
| CD185 | Human | J252D4 | PE-Cy7 | Trafficking | B memory, DNB | Med |
| CD24 | Human | ML5 | Brilliant Ultraviolet 395 | n/a | Transitional B, B memory | High |
| CD38 | Human | HIT2 | Brilliant Ultraviolet 661 | Differentiation B cells | Plasma Cell, Transitional B | Med |
| CD183 | Human | G025H7 | Brilliant Violet 421 | Trafficking | Plasma Cell | Med |
| CD14 | Human | MfP9 | Brilliant Violet 510 | Exclusion | High | |
| CD43 | Human | 1G10 | Brilliant Violet 605 | Differentiation B cells | B memory, Plasma cell | Low |
| CD39 | Human | TU66 | Brilliant Violet 711 | Inhibitory/exhaustion marker | B memory | Low |
| CD32 | Human | 3D3 | Brilliant Violet 750 | Inhibitory/exhaustion marker | B memory | Med |
| IgM | Human | MHM-88 | Alexa Fluor 700 | Differentiation B cells | B memory | Med |
| CD3 | Human | UCHT1 | Brilliant Violet 510 | Exclusion | High | |
| CD5 | Human | CD5-5D7 | PE-Cy5.5 | Differentiation B cells | Transitional B | High |
| CD21 | Human | B-ly4 | Brilliant Ultraviolet 496 | Differentiation B cells | B memory | Low |
| CD19 | Human | HIB19 | APC-H7 | Lineage B cells | Transitional B, B memory, Plasma cell, DNB | Low |
| CD27 | Human | M-T271 | PE-CF594 | Differentiation B cells | B memory, Plasma Cell | Med |
| CD22 | Human | HIB22 | Brilliant Ultraviolet 805 | Inhibitory/exhaustion marker | DNB | Med |
| CD20 | Human | 2H7 | Brilliant Violet 786 | Lineage B cells | B memory, Transitional B, DNB | Med |
| CD272 | Human | J168-540 | Brilliant Blue 700 | Inhibitory/exhaustion marker | B memory | Med |
