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* These authors contributed equally
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.
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.
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....
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.......
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.......
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.......
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.
....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 | BDÂ | 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 | |
CD23Â | 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 |
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