In this invited article, we explain technical aspects of the lymphocytic choriomeningitis virus (LCMV) system, providing an update of a prior contribution by Matthias von Herrath and J. Lindsay Whitton. We provide an explanation of the LCMV infection models, highlighting the importance of selecting an appropriate route and viral strain. We also describe how to quantify virus‐specific immune responses, followed by an explanation of useful transgenic systems. Specifically, our article will focus on the following protocols. © 2020 Wiley Periodicals LLC.
Antigen‐specific memory B cell (MBC) populations mediate the rapid, strong, and high‐affinity secondary antibody responses that play a key role in combating infection and generating protective responses to vaccination. Recently, cell staining with fluorochrome‐labeled antigens together with sequencing methods such as Drop‐seq and CITE‐seq have provided information on the specificity, phenotype, and transcriptome of single MBCs. However, characterization of MBCs at the level of antigen‐reactive populations remains an important tool for assessing an individual's B cell immunity and responses to antigen exposure. This is readily performed using a long‐established method based on in vitro polyclonal stimulation of MBCs to induce division and differentiation into antibody‐secreting cells (ASCs). Post‐stimulation antigen‐specific measurement of the MBC‐derived ASCs (or the secreted antibodies) indicates the size of precursor MBC populations. Additional information about the character of antigen‐reactive MBC populations is provided by analysis of MBC‐derived antibodies of particular specificities for binding avidity and functionality. This article outlines a simple and reliable strategy for efficient in vitro MBC stimulation and use of the ELISpot assay as a post‐stimulation readout to determine the size of antigen‐specific MBC populations. Other applications of the in vitro stimulation technique for MBC analysis are discussed. The following protocols are included. © 2020 Wiley Periodicals LLC
- NSF-PAR ID:
- 10238193
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Current Protocols in Immunology
- Volume:
- 131
- Issue:
- 1
- ISSN:
- 1934-3671
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Basic Protocol 1 : Isolation of bone marrow progenitor cellsBasic Protocol 2 : In vitro differentiation of dendritic cells with GM‐CSFSupport Protocol 1 : Preparation of conditioned medium from GM‐CSF producing J558L cellsBasic Protocol 3 : In vitro differentiation of dendritic cells with Flt3LSupport Protocol 2 : Preparation of Flt3L containing medium from B16‐Flt3L cellsBasic Protocol 4 : Expansion of cDC1s in vivo for use in ex vivo experimentsBasic Protocol 5 : Characterizing resting and activated dendritic cellsBasic Protocol 6 : Dendritic cell stimulation, antigenic cargo, and fixationSupport Protocol 3 : Preparation of model antigen coated microbeadsSupport Protocol 4 : Preparation of apoptotic cellsSupport Protocol 5 : Preparation of recombinant bacteriaBasic Protocol 7 : Immunocytochemistry immunofluorescence (ICC/IF)Support Protocol 6 : Preparation of Alcian blue‐coated coverslipsBasic Protocol 8 : CD8+T cell activation to assess cross‐presentationSupport Protocol 7 : Isolation and labeling of CD8+T cells with CFSE -
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