Sign In

A subscription to JoVE is required to view this content. Sign in or start your free trial.

In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

We describe herein a simple analysis of the heterogeneity of the murine immune B cell compartment in the peritoneum, spleen, and bone marrow tissues by flow cytometry. The protocol can be adapted and extended to other mouse tissues.

Abstract

Extensive studies have characterized the development and differentiation of murine B cells in secondary lymphoid organs. Antibodies secreted by B cells have been isolated and developed into well-established therapeutics. Validation of murine B cell development, in the context of autoimmune prone mice, or in mice with modified immune systems, is a crucial component of developing or testing therapeutic agents in mice and is an appropriate use of flow cytometry. Well established B cell flow cytometric parameters can be used to evaluate B cell development in the murine peritoneum, bone marrow, and spleen, but a number of best practices must be adhered to. In addition, flow cytometric analysis of B cell compartments should also complement additional readouts of B cell development. Data generated using this technique can further our understanding of wild type, autoimmune prone mouse models as well as humanized mice that can be used to generate antibody or antibody-like molecules as therapeutics.

Introduction

Monoclonal antibodies have increasingly become the choice therapy for many human diseases as they become part of mainstream medicine1,2. We have previously described genetically engineered mice which efficiently produce antibodies harboring fully human variable regions with mouse IgH constants3,4. Most recently, we have described genetically engineered mice that produce antibody-like molecules that have distinct antigen-binding5. Antibodies are secreted by B cells and form the basis of adaptive humoral immunity. There are two di....

Protocol

All mouse studies were overseen and approved by Regeneron's Institutional Animal Care and Use Committee (IACUC). The experiment was conducted on tissues from three C57BL/6J female mice (17 weeks of age) from Jackson Laboratories. Titrate all antibodies prior to starting the experiment to determine ideal concentration. When using compensation beads for single-color compensation, ensure they stain as bright or brighter than your samples. Keep all buffers, antibodies, and cells on ice or at 4 °C. After the addition.......

Representative Results

Here we present the gating strategy for characterizing B cell development in mouse peritoneum, BM and spleen. The basis of the analysis is formed around the concept of staining with viability dye, then gating out doublets based on the Forward-Scatter-Area (FSC-A) and Forward-Scatter-Height (FSC-H), and finally gating out debris by selecting cells according to their FSC-A and Side-Scatter-Area (SSC-A) characteristics, referred to here as the size gate, which are reflective of relative cell.......

Discussion

Flow cytometric analysis of lymphoid and non-lymphoid tissues has enabled simultaneous identification and enumeration of B cell sub-populations in mice and humans since the 1980's. It has been used as a measure of humoral immunity and can be applied further to evaluate B cell functionality. This method takes advantage of reagent availability to assess different stages of B cell maturation in mice and humans, by way of simultaneous analysis of multiple parameters enabling the assessment of B cell heterogeneity, even i.......

Acknowledgements

We thank Matthew Sleeman for critical reading of the manuscript. We also thank the Vivarium Operations and Flow Cytometry Core departments at Regeneron for supporting this research.

....

Materials

NameCompanyCatalog NumberComments
0.5 mL safe-lock Eppendorf tubesEppendorf 223636110.5 mL microcentrifuge tube
1.5mL Eppendorf tubes Eppendorf 223641111.5 mL microcentrifuge tube
15 mL Falcon tubes Corning 35209715 mL conical tube
18 gauge needleBD305196
25 gauge needleBD 305124
3 mL syringeBD309657
70 mM MACS SmartStrainer Miltenyi Biotec 130-110-916 70 mM cell strainer
96 well U bottom plate VWR10861-564
ACK lysis buffer GIBCO A1049201red blood cell lysis buffer
Acroprep Advance 96 Well Filter PlatePall Corporation8027filter plate
B220eBiosciences17-0452-82
BD CompBead Anti-Mouse Ig/κBD552843compensation beads
BD CompBead Anti-Rat Ig/κBD552844compensation beads
Bovine Serum AlbuminSigma-Aldrich A8577BSA
BP-1BD740882
Brilliant Stain BufferBD566349brilliant stain buffer
C-KitBD564011
CD11bBD563168
CD11bBioLegend101222
CD19BD560143
CD21/35BD562756
CD23 BD740216
CD24 (HSA)BioLegend138504
CD3BD561388
CD3BioLegend100214
CD43BD553270
CD43BioLegend121206
CD5BD563194
CD93BD740750
CD93BioLegend136504
DPBS (1x)ThermoFisher14190-144DPBS
eBioscience Fixable Viability Dye eFluor 506ThermoFisher65-0866-14viability dye
Extended Fine Tip Transfer PipetteSamco233disposable transfer pipette
FACSymphony A3 flow cytometerBDcustom orderflow cytometer
Fc Block, CD16/CD32 (2.4G2)BD553142Fc block
FlowJoFlowjoflow cytometer analysis software
gentleMACS C Tubes Miltenyi Biotec 130-096-334automated dissociation tube 
gentleMACS Octo Dissociator with Heaters Miltenyi Biotec 130-095-937tissue dissociator instrument
GR1 (Ly6C/6G)BioLegend108422
IgDBioLegend405710
IgMeBiosciences25-5790-82
KappaBD550003
LambdaBioLegend407308
paraformaldehyde, 32% SolutionElectron Microscopy Sciences15714
Ter119BioLegend116220
True-Stain Monocyte BlockerBioLegend426103monocyte blocker
UltraPure EDTA, pH 8.0ThermoFisher15575038EDTA
Vi-CELL XRBeckman Coulter731050cell counter instrument 

References

  1. Shepard, H. M., Philips, G. L., Thanos, D., Feldman, M. Developments in therapy with monoclonal antibodies and related proteins. Clinical Medicine. 17 (3), 220 (2017).
  2. Ecker, D. M., Jones, S. D., Levine, H. L. The therapeutic monoclonal a....

Explore More Articles

Flow CytometryB Cell DevelopmentMurine ModelsAutoimmunityAntibody TherapeuticsFluorophoresPhenotypingGenetic ManipulationBone MarrowSpleenViability DyeSingle Color Compensation

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved