Protocol for in vitro BCR-mediated plasma cell differentiation and purification of chromatin-associated proteins

• Published in: STAR protocols Vol. 2; no. 3; p. 100633
• Main Authors: Ochiai, Kyoko, Shima, Hiroki, Ikura, Tsuyoshi, Franke, Marissa C., Sievert, Evelyn P., Sciammas, Roger, Igarashi, Kazuhiko
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 17-09-2021; Elsevier
• Subjects: Antibody; Cell Biology; Cell Differentiation; Chromatin immunoprecipitation (ChIP); Flow Cytometry/Mass Cytometry; Gene Expression; Immunology; Mass Spectrometry; Molecular Biology; Protocol; Chromatin immunoprecipitation (ChIP); Gene Expression; Immunology; Molecular Biology; Antibody; Flow Cytometry/Mass Cytometry; Mass Spectrometry; Cell Differentiation; Cell Biology
• ISSN: 2666-1667; 2666-1667
• DOI: 10.1016/j.xpro.2021.100633

Molecular-level understanding of plasma cell (PC) differentiation has been modeled using lipopolysaccharide (LPS) stimulation in vitro. However, this system does not involve the B-cell receptor (BCR)—a critical component of B cell biology. Here, we present a protocol for in vitro PC differentiation system dependent on BCR signaling that easily scales up for cell number-demanding applications, including protein complex purification. We describe how to set up this system and detail applications for endogenous complex purification of chromatin-associated proteins.
For further details on the use and execution of this protocol, please refer to Sciammas et al. (2011) and Ochiai et al. (2018, 2020).
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•In vitro plasma cell differentiation system via B-cell receptor signaling•Antibody conjugation of protein A or G beads for immunoprecipitation•Purification of endogenous protein complexes and chromatin-associated proteins•Gel extraction and digestion of proteins in preparation for LC-MS/MS analysis
Molecular-level understanding of plasma cell (PC) differentiation has been modeled using LPS stimulation in vitro. However, this system does not involve the B-cell receptor (BCR)—a critical component of B cell biology. Here, we present a protocol for in vitro PC differentiation system dependent on BCR signaling that easily scales up for cell number-demanding applications, including protein complex purification. We describe how to set up this system and detail applications for endogenous complex purification of chromatin-associated proteins.