Flow Cytometric Characterization of Murine B Cell Development

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 medicine^1,2. We have previously described genetically engineered mice which efficiently produce antibodies harboring fully human variable regions with mouse IgH constants^3,4. 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 of viability dye, perform all steps and incubations at 4°C in either low light or in the dark.

1. Peritoneal cell harv....

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.5 mL safe-lock Eppendorf tubes	Eppendorf	22363611	0.5 mL microcentrifuge tube
1.5mL Eppendorf tubes	Eppendorf	22364111	1.5 mL microcentrifuge tube
15 mL Falcon tubes	Corning	352097	15 mL conical tube
18 gauge needle	BD	305196
25 gauge needle	BD	305124
3 mL syringe	BD	309657
70 mM MACS SmartStrainer	Miltenyi Biotec	130-110-916	70 mM cell strainer
96 well U bottom plate	VWR	10861-564
ACK lysis buffer	GIBCO	A1049201	red blood cell lysis buffer
Acroprep Advance 96 Well Filter Plate	Pall Corporation	8027	filter plate
B220	eBiosciences	17-0452-82
BD CompBead Anti-Mouse Ig/κ	BD	552843	compensation beads
BD CompBead Anti-Rat Ig/κ	BD	552844	compensation beads
Bovine Serum Albumin	Sigma-Aldrich	A8577	BSA
BP-1	BD	740882
Brilliant Stain Buffer	BD	566349	brilliant stain buffer
C-Kit	BD	564011
CD11b	BD	563168
CD11b	BioLegend	101222
CD19	BD	560143
CD21/35	BD	562756
CD23	BD	740216
CD24 (HSA)	BioLegend	138504
CD3	BD	561388
CD3	BioLegend	100214
CD43	BD	553270
CD43	BioLegend	121206
CD5	BD	563194
CD93	BD	740750
CD93	BioLegend	136504
DPBS (1x)	ThermoFisher	14190-144	DPBS
eBioscience Fixable Viability Dye eFluor 506	ThermoFisher	65-0866-14	viability dye
Extended Fine Tip Transfer Pipette	Samco	233	disposable transfer pipette
FACSymphony A3 flow cytometer	BD	custom order	flow cytometer
Fc Block, CD16/CD32 (2.4G2)	BD	553142	Fc block
FlowJo	Flowjo		flow cytometer analysis software
gentleMACS C Tubes	Miltenyi Biotec	130-096-334	automated dissociation tube
gentleMACS Octo Dissociator with Heaters	Miltenyi Biotec	130-095-937	tissue dissociator instrument
GR1 (Ly6C/6G)	BioLegend	108422
IgD	BioLegend	405710
IgM	eBiosciences	25-5790-82
Kappa	BD	550003
Lambda	BioLegend	407308
paraformaldehyde, 32% Solution	Electron Microscopy Sciences	15714
Ter119	BioLegend	116220
True-Stain Monocyte Blocker	BioLegend	426103	monocyte blocker
UltraPure EDTA, pH 8.0	ThermoFisher	15575038	EDTA
Vi-CELL XR	Beckman Coulter	731050	cell counter instrument