Name: flow cytometric characterization of murine b cell development
Uploaded: 2021-01-22T12:30:00
Description: Watch this Scientific Journal Video about Flow Cytometric Characterization of Murine B Cell Development at JoVE.com

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.

All mouse studies were overseen and approved by Regeneron&#39;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 &#176;C. After the addition...

<ol>
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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&#39;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...

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 distinct types of B cells, B-1 and B-2. In mammals, B-1 cells originate in the fetal liver and are enriched in mucosal tissues and the pleural and peritoneal cavities after birth, while B-2 cells originate in the fetal liver prior to birth and thereafter in the bone marrow (BM). B-2 cells are enriched in secondary lymphoid organs including the spleen and blood6,7,8. In the BM, B-2 hematopoietic progenitors start to differentiate to pro-B cells upon the initiation of Ig mu heavy chain rearrangement9,10. Successful rearrangement of Ig heavy chain and its assembly into the pre-B cell receptor (pre-BCR), along with signaling and proliferative expansion, leads to differentiation to pre-B cells. After pre-B cells rearrange their Ig kappa (Ig&#954;), or if unproductive, Ig lambda (Ig&#955;) light chains, they pair with &#956; heavy chain, resulting in surface IgM BCR expression. It is important to point out that IgM surface expression is known to be reduced under conditions of autoreactivity, thus contributing to self tolerance in functionally unresponsive or anergic B cells11,12. Immature B cells then enter a transitional stage, where they begin to co-express IgD and migrate from the BM to the spleen. In the spleen, IgD expression increases further and the cells mature into a second stage of transitional B cells, followed by completion of their maturation status and development into either marginal zone (MZ) or follicular (Fol) cells13,14,15. In adult mice, in a non-diseased setting, the number of mature B cells remains constant despite 10-20 million immature B cells being generated daily in the BM. Of these, only three percent enter the pool of mature B cells. The size of the peripheral B cell compartment is constrained by cell death, due in part to several factors including self-reactivity and incomplete maturation16,17,18. Flow cytometric analysis has been extensively used to characterize and enumerate many immune cell sub-compartments in humans and mice. While there are some similarities between human and murine B cell compartments, this protocol applies only to the analysis of murine B cells. This protocol was developed with the purpose of phenotyping genetically engineered mice, to determine whether genetic manipulation would alter B cell development. Flow cytometry has also been hugely popular in many additional applications, including in measuring cell activation, function, proliferation, cycle analysis, DNA content analysis, apoptosis and cell sorting 19,20.
Flow cytometry is the tool of choice to characterize various lymphocyte compartments in mice and humans, including in complex organs such as the spleen, BM and blood. Due to widely available mouse-specific antibody reagents for flow cytometry, this technique can be used to investigate not only cell surface proteins but also intracellular phosphoproteins and cytokines, as well as functional readouts21. Herein we demonstrate how flow cytometry reagents can be used to identify B cells subsets as they mature and differentiate in secondary lymphoid organs. After optimization of staining conditions, sample handling, correct instrument set up and data acquisition, and finally data analysis, a protocol for comprehensive flow cytometric analysis of the B cell compartment in mice can be utilized. Such comprehensive analysis is based on a decades old nomenclature devised by Hardy and colleagues, where developing BM B-2 cells can be divided into different fractions (Fraction) depending on their expression of B220, CD43, BP-1, CD24, IgM and IgD22. Hardy et al., showed that B220+ CD43 BM B cells can be subdivided into four subsets (Fraction A-C&#39;) on the basis of BP-1 and CD24 (30F1) expression, while B220+ CD43-(dim to neg) BM B cells can be resolved into three subsets (Fraction D-F) based on differential expression of IgD and surface IgM23. Fraction A (pre-pro-B cells) are defined as BP-1- CD24 (30F1)-, Fraction B (early pro-B cells) are defined as BP-1- CD24 (30F1)+, Fraction C (late pro-B cells) are defined as BP-1+ CD24 (30F1)+, and Fraction C&#39; (early pre-B cells) are defined as BP-1+ and CD24high. Furthermore, Fraction D (pre-B cells) are defined as B220+ CD43- IgM- B cells, and Fraction E (newly generated B cells, combination of immature and transitional) are defined as B220+ CD43- IgM+ B cells and Fraction F (mature, recirculting B cells) are defined as B220high CD43- IgM+ B cells. In contrast, the majority of na&#239;ve B cells found in the spleen can be divided into mature (B220+ CD93-) B cells and transitional (T1, T2, T3) cells depending on expression of CD93, CD23 and IgM. Mature B cells can be resolved into marginal zone and follicular subsets based on expression of IgM and CD21/CD35, and follicular subsets can be further divided into mature follicular type I and follicular type II B cell subsets depending on the level of their IgM and IgD surface expression24. These splenic B cell populations express predominantly Ig&#954; light chain. Finally, B-1 B cell populations, which originate in the fetal liver and are mainly found in the peritoneal and pleural cavities of adult mice, have been described in the literature. These peritoneal B cells can be distinguished from the previously described B-2 B cells by their lack of CD23 expression. They are then further subdivided into B-1a or B-1b populations, with the former defined by the presence of CD5 and the latter by its absence25. B-1 cell progenitors are abundant in the fetal liver, but are not found in adult BM. While B-1a and B-1b cells originate from different progenitors, they both seed the peritoneal and pleural cavities24. In contrast to B-2 cells, B-1 cells are uniquely capable of self-renewal and are responsible for production of natural IgM antibodies.
Defects in B cell development can arise in many instances, including deficiencies in the components of the BCR26,27, perturbations of signaling molecules that impact BCR signaling strength14,28,29, or disruption of cytokines that modulate B cell survival30,31. Flow cytometry analysis of the lymphoid compartments has contributed to the characterization of the B cell development blocks in these mice and many others. One advantage of flow cytometric analysis of lymphoid compartments is that it offers the ability to make measurements on individual cells obtained from live dissociated tissue. The availability of reagents in an ever-expanding range of fluorophores allows for the simultaneous analysis of multiple parameters and enables the assessment of B cell heterogeneity. Furthermore, enumeration of B cells by flow cytometric analysis complements other immunological assays such as immunohistochemistry methods that visualize cell localization within lymphoid organs, detection of circulating antibody levels as a measure of humoral immunity, as well as two photon microscopy to measure B cell responses in real space and time32.

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	<tbody>
		<tr>
			<td>0.5 mL safe-lock Eppendorf tubes</td>
			<td>Eppendorf&#160;</td>
			<td>22363611</td>
			<td>0.5 mL microcentrifuge tube</td>
		</tr>
		<tr>
			<td>1.5mL Eppendorf tubes&#160;</td>
			<td>Eppendorf&#160;</td>
			<td>22364111</td>
			<td>1.5 mL microcentrifuge tube</td>
		</tr>
		<tr>
			<td>15 mL Falcon tubes&#160;</td>
			<td>Corning&#160;</td>
			<td>352097</td>
			<td>15 mL conical tube</td>
		</tr>
		<tr>
			<td>18 gauge needle</td>
			<td>BD</td>
			<td>305196</td>
			<td></td>
		</tr>
		<tr>
			<td>25 gauge needle</td>
			<td>BD&#160;</td>
			<td>305124</td>
			<td></td>
		</tr>
		<tr>
			<td>3 mL syringe</td>
			<td>BD</td>
			<td>309657</td>
			<td></td>
		</tr>
		<tr>
			<td>70 mM MACS SmartStrainer&#160;</td>
			<td>Miltenyi Biotec&#160;</td>
			<td>130-110-916&#160;</td>
			<td>70 mM cell strainer</td>
		</tr>
		<tr>
			<td>96 well U bottom plate&#160;</td>
			<td>VWR</td>
			<td>10861-564</td>
			<td></td>
		</tr>
		<tr>
			<td>ACK lysis buffer&#160;</td>
			<td>GIBCO&#160;</td>
			<td>A1049201</td>
			<td>red blood cell lysis buffer</td>
		</tr>
		<tr>
			<td>Acroprep Advance 96 Well Filter Plate</td>
			<td>Pall Corporation</td>
			<td>8027</td>
			<td>filter plate</td>
		</tr>
		<tr>
			<td>B220</td>
			<td>eBiosciences</td>
			<td>17-0452-82</td>
			<td></td>
		</tr>
		<tr>
			<td>BD CompBead Anti-Mouse Ig/&#954;</td>
			<td>BD</td>
			<td>552843</td>
			<td>compensation beads</td>
		</tr>
		<tr>
			<td>BD CompBead Anti-Rat Ig/&#954;</td>
			<td>BD</td>
			<td>552844</td>
			<td>compensation beads</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>A8577</td>
			<td>BSA</td>
		</tr>
		<tr>
			<td>BP-1</td>
			<td>BD</td>
			<td>740882</td>
			<td></td>
		</tr>
		<tr>
			<td>Brilliant Stain Buffer</td>
			<td>BD</td>
			<td>566349</td>
			<td>brilliant stain buffer</td>
		</tr>
		<tr>
			<td>C-Kit</td>
			<td>BD</td>
			<td>564011</td>
			<td></td>
		</tr>
		<tr>
			<td>CD11b</td>
			<td>BD</td>
			<td>563168</td>
			<td></td>
		</tr>
		<tr>
			<td>CD11b</td>
			<td>BioLegend</td>
			<td>101222</td>
			<td></td>
		</tr>
		<tr>
			<td>CD19</td>
			<td>BD</td>
			<td>560143</td>
			<td></td>
		</tr>
		<tr>
			<td>CD21/35</td>
			<td>BD</td>
			<td>562756</td>
			<td></td>
		</tr>
		<tr>
			<td>CD23&#160;</td>
			<td>BD</td>
			<td>740216</td>
			<td></td>
		</tr>
		<tr>
			<td>CD24 (HSA)</td>
			<td>BioLegend</td>
			<td>138504</td>
			<td></td>
		</tr>
		<tr>
			<td>CD3</td>
			<td>BD</td>
			<td>561388</td>
			<td></td>
		</tr>
		<tr>
			<td>CD3</td>
			<td>BioLegend</td>
			<td>100214</td>
			<td></td>
		</tr>
		<tr>
			<td>CD43</td>
			<td>BD</td>
			<td>553270</td>
			<td></td>
		</tr>
		<tr>
			<td>CD43</td>
			<td>BioLegend</td>
			<td>121206</td>
			<td></td>
		</tr>
		<tr>
			<td>CD5</td>
			<td>BD</td>
			<td>563194</td>
			<td></td>
		</tr>
		<tr>
			<td>CD93</td>
			<td>BD</td>
			<td>740750</td>
			<td></td>
		</tr>
		<tr>
			<td>CD93</td>
			<td>BioLegend</td>
			<td>136504</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS (1x)</td>
			<td>ThermoFisher</td>
			<td>14190-144</td>
			<td>DPBS</td>
		</tr>
		<tr>
			<td>eBioscience Fixable Viability Dye eFluor 506</td>
			<td>ThermoFisher</td>
			<td>65-0866-14</td>
			<td>viability dye</td>
		</tr>
		<tr>
			<td>Extended Fine Tip Transfer Pipette</td>
			<td>Samco</td>
			<td>233</td>
			<td>disposable transfer pipette</td>
		</tr>
		<tr>
			<td>FACSymphony A3 flow cytometer</td>
			<td>BD</td>
			<td>custom order</td>
			<td>flow cytometer</td>
		</tr>
		<tr>
			<td>Fc Block, CD16/CD32 (2.4G2)</td>
			<td>BD</td>
			<td>553142</td>
			<td>Fc block</td>
		</tr>
		<tr>
			<td>FlowJo</td>
			<td>Flowjo</td>
			<td></td>
			<td>flow cytometer analysis software</td>
		</tr>
		<tr>
			<td>gentleMACS C Tubes&#160;</td>
			<td>Miltenyi Biotec&#160;</td>
			<td>130-096-334</td>
			<td>automated dissociation tube&#160;</td>
		</tr>
		<tr>
			<td>gentleMACS Octo Dissociator with Heaters&#160;</td>
			<td>Miltenyi Biotec&#160;</td>
			<td>130-095-937</td>
			<td>tissue dissociator instrument</td>
		</tr>
		<tr>
			<td>GR1 (Ly6C/6G)</td>
			<td>BioLegend</td>
			<td>108422</td>
			<td></td>
		</tr>
		<tr>
			<td>IgD</td>
			<td>BioLegend</td>
			<td>405710</td>
			<td></td>
		</tr>
		<tr>
			<td>IgM</td>
			<td>eBiosciences</td>
			<td>25-5790-82</td>
			<td></td>
		</tr>
		<tr>
			<td>Kappa</td>
			<td>BD</td>
			<td>550003</td>
			<td></td>
		</tr>
		<tr>
			<td>Lambda</td>
			<td>BioLegend</td>
			<td>407308</td>
			<td></td>
		</tr>
		<tr>
			<td>paraformaldehyde, 32% Solution</td>
			<td>Electron Microscopy Sciences</td>
			<td>15714</td>
			<td></td>
		</tr>
		<tr>
			<td>Ter119</td>
			<td>BioLegend</td>
			<td>116220</td>
			<td></td>
		</tr>
		<tr>
			<td>True-Stain Monocyte Blocker</td>
			<td>BioLegend</td>
			<td>426103</td>
			<td>monocyte blocker</td>
		</tr>
		<tr>
			<td>UltraPure EDTA, pH 8.0</td>
			<td>ThermoFisher</td>
			<td>15575038</td>
			<td>EDTA</td>
		</tr>
		<tr>
			<td>Vi-CELL XR</td>
			<td>Beckman Coulter</td>
			<td>731050</td>
			<td>cell counter instrument&#160;</td>
		</tr>
	</tbody>
</table>

flow cytometric characterization of murine b cell development

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.

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...

Watch this Scientific Journal Video about Flow Cytometric Characterization of Murine B Cell Development at JoVE.com

Flow Cytometric Characterization of Murine B Cell Development

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 ...

Data generated using this technique furthers an understanding of autoimmune prone mouse models, as well as humanized mice, that can be used to general antibody or antibody life therapeutics. The availability of reagents with an ever-expanding range of fluorophores, allows for the simultaneous analysis of multiple parameters on individual cells and enables the assessment of B cell heterogeneity. This protocol was developed with the purpose of phenotyping genetically engineered mice.to determine whether genetic manipulation would alter B cell development. Hi, I'm Faith Harris. I'll be demonstrating the procedure today.Begin by aliquoting a million of each cell type from each animal into a 96-well U-bottom plate. Make sure sufficient Wells are available for all samples and controls, including full stain, fluorescence-minus-one samples, and unstained samples for each fluorophore. For the bone marrow maturation panel and the spleen maturation panel, aliquot cells into two wells, 1 million cells per well for each full stain sample.For the single color compensation viability controls, add two million cells each of each cell type to individual wells. Centrifuge the plate at 845 x g for two minutes at four degrees Celsius. Decant the supernatant by quickly inverting and flicking the plate over a sink, taking care not to cross-contaminate the wells.Wash the cells twice in 200 microliters of DPBS. Centrifuge and decant the supernatant after each wash. Resuspend the cells in 100 microliters of viability dye diluted in DPBS at the ratio of one to 1000, avoiding the cells used for single color compensation.For each stain set, leave several unstained wells for a completely unstained sample and other controls you might need as well as an additional unstained well for viability FMO control. Add PBS to these wells. Resuspend the cells of single color compensation viability controls in 200 microliters of diluted viability dye.Transfer 100 microliters of these cells to a 1.5 milliliter microcentrifuge tube and heat it for five minutes at 65 degrees Celsius, then transfer the heated cells back to the original well with the remaining live cells. Incubate the cells at four degrees Celsius for 30 minutes protected from light. After the incubation, centrifuge the cells and decant the supernatant by flicking or inverting the plate without cross-contaminating the other wells.Wash the cells by resuspending in 200 microliters of DPBS, then centrifuge and decant the supernatant as before. Repeat the DPBS wash again. Add microliters of FC block diluted with stain buffer at a ratio of one to 50 to get a final concentration of 10 micrograms per milliliter.For peritoneal cells, add five microliters of monocyte blocker to reduce the nonspecific staining. Then, incubate the cells at four degrees Celsius for 15 minutes, protected from light. Prepare full stain master mixes and FMOs in the stain buffer for a final volume of 100 microliters per million cells using the antibody tables in the text manuscript.Without removing FC block, add 100 microliters of full stain mixes and FMOs to selected wells. Prepare single color compensation controls for each antibody and a stain set. Follow the manufacturer's directions if using compensation beads.Incubate the cells and the beads at four degrees Celsius for 30 minutes, protected from light. Then, centrifuge and decant the supernatant by inverting and flicking the plate. Wash the cells and beads in 200 microliters of stain buffer three times, centrifuging and decanting the supernatant each time.Resuspend the cells and beads in 200 microliters of 2%paraformaldehyde in DPBS to fix the samples for analysis. Incubate the cell and beads at four degrees Celsius for 30 minutes, protected from light, then centrifuge and decant the supernatant by inverting or flicking the plate. Resuspend the cells and beads in 200 microliters of stain buffer.Place a filter plate over a clean 96-well U-bottom plate and transfer each sample to a well of the filter plate using a multi-pipette. Centrifuge the filter plate and decant the supernatant. For the bone marrow and spleen maturation panels, resuspend the fully stained cells in 100 microliters of stain buffer.Combine the two wells for each animal into one well. Resuspend the remaining panels, FMOs, and controls in 200 microliters of stain buffer. Incubate fixed cells and beads at four degrees Celsius overnight, protected from light.Record compensation controls for each stain panel using the prepared single stain compensations and set positive and negative gates for each sample. Calculate the compensation matrix using the software. Start acquiring the first sample, making sure that the gates are set appropriately.Set the machine to run and record the various cell events for each sample as mentioned in the text manuscript. Proceed with data analysis using flow cytometry analysis software, following the gating strategies in the text manuscript. Flow cytometric analysis for characterization of peritoneal B cells in the peritoneum shows the frequencies of viable peritoneal cells, total B cells, B-1 and B-2 subsets, as well as B-1a and B-1b cells in mice.When bone marrow B cells were analyzed, frequencies of viable cells, total B cells, Fraction A, pre-pro-B cells, Fraction B, Fraction C, Fraction C'Fraction D, immature Fraction E, transitional Fraction E, and fraction F B cells in mice were determined. Analysis of splenic B cells shows the frequencies of viable spleen cells, total B cells, transitional B cells, T1, T2, T3 cells, mature B cells, follicular I cells, follicular II cells, precursor and mature marginal zone cells, and B-1 cells in mice. The frequencies of immunoglobulin kappa and immunoglobulin lambda B cells in mice were determined with flow cytometric analysis of the spleen.When performing this protocol, you need to resolve signal from one detector bleeding over into the next using compensation controls. These controls could either be cell singly stained or with commercially available compensation beads. Additional assays can be used in conjunction with flow cytometry, like immunohistochemistry to visualize cell localization within lymphoid organs, as well as two photon microscopy to analyze B cell responses in real space and time.Flow cytometry analysis of the B cell compartments allows for characterization of phenotypes associated with BCR and related deficiencies, perturbations of BCR signaling molecules, or disruption of cytokines that modulate B cell survival.

Research

JoVE Journal

Immunology and Infection

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A Murine Model of Group B Streptococcus Vaginal Colonization

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