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 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 blood^6,7,8. In the BM, B-2 hematopoietic progenitors start to differentiate to pro-B cells upon the initiation of Ig mu heavy chain rearrangement^9,10. 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 (Igκ), or if unproductive, Ig lambda (Igλ) light chains, they pair with μ 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 cells^11,12. 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) cells^13,14,15. 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 maturation^16,17,18. 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') 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' (early pre-B cells) are defined as BP-1⁺ and CD24^high. 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 B220^high CD43^- IgM⁺ B cells. In contrast, the majority of naïve 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 Igκ 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 BCR^26,27, perturbations of signaling molecules that impact BCR signaling strength^14,28,29, or disruption of cytokines that modulate B cell survival^30,31. 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.

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 harvest and single cell isolation

1. Euthanize the mouse using CO₂ or according to approved protocol_.
2. Lay the mouse on its back, spray with 70% ethanol, and cut outer abdominal skin with scissors, being careful not to cut the peritoneum.
3. Inject 3 mL of ice-cold wash buffer (0.5% bovine serum albumin (BSA) in DPBS [vol/vol]) into the peritoneal cavity with a 3 mL syringe fitted with a 25 gauge needle.
4. Gently massage the peritoneum with fingertips.
5. Repeat steps 1.3 and 1.4.
6. Insert a 3 mL syringe fitted with an 18 G needle through the peritoneum, being careful to avoid organs and fat.
7. Extract the wash buffer, now containing peritoneal cells, and transfer to 15 mL conical tube on ice.
8. Repeat steps 1.3 and 1.4.
9. Cut a small hole in the peritoneum while holding up with tweezers.
10. Insert a disposable transfer pipette into the hole and collect the remaining wash buffer, once again avoiding fat and organs.
11. Transfer the collected remaining peritoneal cells to the 15 mL conical tube on ice.
    NOTE: Discard sample if blood contamination is evident.
12. Incubate the cells on ice until spleen and bone extraction are complete.
13. Centrifuge the cells at 300 x g for 8 min at 4 ˚C. Aspirate the supernatant.
14. Resuspend the cell pellet in 1 mL of wash buffer.
15. Filter the cells through a 70 µM cell strainer into a clean 15 mL conical tube on ice.
16. Determine the cell concentration using a cell counter instrument or hemocytometer.

2. Spleen harvest and single cell isolation

1. Lay the mouse on its belly and cut through the peritoneum on the left backside using clean scissors. Cut out the spleen, removing fat and connective tissue.
2. Transfer the spleen to a 1.5 mL microcentrifuge tube containing 1 mL of wash buffer on ice.
3. Incubate the spleen on ice until bone extraction is complete.
4. Move the spleen to automated dissociation tube with 5 mL of red blood cell lysis buffer. Place the tube on the tissue dissociator instrument and dissociate for 60 s to create a single cell suspension.
   NOTE: It is also permissible to use other routine methods of obtaining single-cell spleen suspensions such as smashing between frosted glass slides in wash buffer. If another method of dissociation is used, follow the dissociation with centrifugation, aspiration, and then resuspension in 5 mL of red blood cell lysis buffer before continuing to step 2.5.
5. Incubate the cells at room temperature for 3 min.
6. Add 10 mL of 4 °C wash buffer containing 2mM EDTA.
7. Transfer to a clean 15 mL conical tube.
8. Centrifuge the cells at 300 x g for 8 min at 4 °C. Aspirate the supernatant.
9. Resuspend the cell pellet in 5 mL of 4 °C wash buffer.
10. Filter the cells through a 70 µM cell strainer into a clean 15 mL conical tube on ice.
11. Determine the cell concentration using a cell counter instrument or hemocytometer.

3. BM harvest and single cell isolation

1. Remove the skin from the lower half of the mouse body. Trim the excess muscle from leg. Remove the entire leg with scissors, being careful not to cut the femur. Clean the femur and tibia by removing remaining muscle, fat, and feet.
2. Transfer the bones to a 1.5 mL microcentrifuge tube containing 1 mL of wash buffer on ice.
3. Perforate the bottom of a 0.5 mL microcentrifuge tube, leaving a hole small enough for leg bones not to protrude. Insert the 0.5 mL tube into a clean 1.5 mL microcentrifuge tube. Snip off the end of the femur and tibia proximal to the knee and place the cut ends facing down into the 0.5 mL tube.
4. Centrifuge the cells at 6,780 x g for 2 min at 4 °C.
5. Resuspend the cell pellet in 1 mL of red blood cell lysis buffer and transfer to a 15 mL conical tube containing an additional 3 mL of red blood cell lysis buffer.
6. Incubate at room temperature for 3 min.
7. Add 10 mL of 4 °C wash buffer containing 2mM EDTA.
8. Centrifuge the cells at 300 x g for 8 min at 4 °C. Aspirate the supernatant.
9. Resuspend the cell pellet in 3 mL of 4 °C wash buffer.
10. Filter cells through a 70 µM cell strainer into a clean 15 mL conical tube on ice.
11. Determine the cell concentration using a cell counter instrument or hemocytometer.

4. Stain cells and prepare compensation

1. Aliquot 10⁶ cells of each cell type from each animal to a 96 well U bottom plate.
   1. Make sure to include enough wells for all samples and controls, including full stain, fluorescence-minus-one (FMO), unstained, and finally the optional single-color compensation for each fluorophore used.
   2. For the BM maturation panel and the spleen maturation panel, aliquot cells into 2 wells, 10⁶ cells per well, for each full stain sample. For the single-color compensation viability controls, add 2 x 10⁶ cells of each cell type to individual wells.
2. Centrifuge the plate at 845 x g for 2 min at 4 °C. Decant the supernatant by quickly inverting and flicking the plate over a sink, being careful not to cross-contaminate wells.
3. Resuspend the cells in 200 µL of DPBS (without BSA or FBS). This step is important to remove protein before staining with amine-reactive viability dye.
4. Repeat steps 4.2 and 4.3.
5. Repeat step 4.2.
6. Resuspend the cells in 100 µL viability dye diluted 1:1,000 in DPBS.
   ​NOTE: If using cells for single-color compensation, do not add viability dye to those wells.
   1. For each stain set, leave several unstained wells for a completely unstained sample and any other controls you might need.
   2. For each stain set, leave an additional unstained well for the viability FMO control.
   3. For the single-color viability compensation controls: Resuspend the 2 x 10⁶ cells, aliquoted in step 4.1, in 200 µL of diluted viability dye. Transfer 100 µL of cells to a 1.5 mL microcentrifuge tube, heat cells for 5 min at 65 °C, and transfer the 100 µL of heat-killed cells back to the original well with the 100 µL remaining live cells.
7. Incubate cells at 4 °C, protected from light, for 30 min.
8. Centrifuge the plate at 845 x g for 2 min at 4 °C. Decant the supernatant by quickly inverting and flicking the plate over a sink, being careful not to cross-contaminate wells.
9. Resuspend the cells in 200 µL of DPBS (without BSA or FBS).
10. Repeat steps 4.8 and 4.9.
11. Repeat step 4.8.
12. Resuspend the cells in 50 µL of Fc block diluted 1:50 (final concentration=10 µg/mL) in stain buffer (0.5% BSA in DPBS [vol/vol]).
    1. For peritoneal cells - also add 5 µL of monocyte blocker to reduce non-specific staining.
13. Incubate the cells at 4 °C, protected from light, for 15 min.
14. Prepare full stain master mixes and FMOs in stain buffer for a final volume of 100 µl per 10⁶ cells.Refer to Table 1-Table 4 for the antibody lists.
    ​NOTE: FMOs are made by including all antibodies in a stain set except one. Prepare an FMO for each antibody in a stain set. When a stain set contains multiple brilliant dyes, substitute 50 µL of brilliant stain buffer for stain buffer per sample
15. Without removing Fc block, add 100 µL of full stain mixes and FMOs to selected wells.
16. Prepare single-color compensation controls for each antibody in a stain set.
    1. If using compensation beads follow the manufacture's directions for use.
    2. If using cells, add titrated antibody to 10⁶ cells, reserved previously in step 4.6.1 without viability dye, in 100 µL stain buffer. If all cells in the sample are positive for a particular marker, set aside unstained cells to be used when acquiring compensation data on the flow cytometer.
17. Incubate the cells and beads at 4 °C, protected from light, for 30 min.
18. Centrifuge the plate at 845 x g for 2 min at 4 °C. Decant the supernatant by quickly inverting and flicking the plate over a sink, being careful not to cross-contaminate wells.
19. Resuspend the cells and beads in 200 µL of stain buffer.
20. Repeat steps 4.18 and 4.19 two times.
21. Repeat step 4.18.
22. To fix the samples for analysis within 48 h, resuspend cells and beads in 200 µL of 2% paraformaldehyde in DPBS.
    CAUTION: Parafomaldehyde is a serious health hazard and flammable. Refer to the Safty Data Sheet before use.
23. Incubate the cells and beads at 4 °C, protected from light, for 30 min.
24. Repeat steps 4.18 and 4.19 two times.
25. Place a filter plate over a clean 96 well U-bottom plate. Using a multi-pipette, transfer each sample to a well of the filter plate.
26. Centrifuge the filter plate-96 well U-bottom plate setup at 845 x g for 2 min at 4 °C. Remove the filter plate and decant the supernatant by quickly inverting and flicking the plate over a sink, being careful not to cross-contaminate wells.
27. For the BM and spleen maturation panels resuspend the fully stained cells in 100 µL of stain buffer. Combine the 2 wells for each animal into 1 well. Resuspend the remaining panels, FMOs, and controls in 200 µL of stain buffer.
28. Incubate fixed cells and beads at 4 °C, protected from light, overnight.

5. Flow cytometric data acquisition

1. Initialize and QC the flow cytometer as per manufacturer instructions.
2. Load the template specific for each panel.
3. Prior to recording data, ensure all events for each sample are on scale and visible on the dot plots.
4. Record compensation controls for each stain panel using single stain compensations prepared in step 4.16. Set positive and negative gates for each sample. Have the software calculate the compensation matrix.
5. Start acquiring the first sample, and ensure gates are set appropriately.
6. Set the machine to record at least 50,000 B cell events for the peritoneal B cell panel and spleen Igκ and Igλ panel; 150,000 B cell events for the BM maturation panel; and 300,000 B cell events for the spleen maturation panel.
7. For each stain panel, run and record the fully stained samples for each animal, an unstained sample, and the FMOs.

6. Analyze data

1. Proceed with data analysis using flow cytometry analysis software. Follow gating strategies outlined in Figure 1, Figure 2, Figure 3, Figure 4.

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 size and cell granularity, before gating on population of interest.

Flow cytometric analysis of peritoneal B cells 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 C57BL/6J mice (Figure 1), using a staining panel outlined in Table 1. Average absolute cell number of these frequencies are shown in Table 5. Perturbations in B-1 cells could be delineated by distribution of cell subsets, either by cell frequency or absolute cells number per mouse.

Flow cytometric analysis of BM B cells shows the frequencies of viable BM cells, total B cells, Fraction A (pre-pro-B cells and contaminating lymphocytes), pre-pro-B cells, Fraction B, Fraction C, Fraction C', Fraction D, immature (subset in Fraction E), transitional (subset in Fraction E), and Fraction F B cells in C57BL/6J mice (Figure 2), using a staining panel outlined in Table 2. Average absolute cell number of these frequencies are shown in Table 6. Perturbations in BM B cells could be delineated by distribution of cell subsets, either by cell frequency or absolute cells number per leg(s).

Flow cytometric 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 (Fol I), follicular II (Fol II) cells, marginal zone (MZ) precursor cells, mature MZ cells, and B-1 cells in C57BL/6J mice (Figure 3), using a staining panel outlined in Table 3. Average absolute cell number of these frequencies are shown in Table 7. Perturbations in splenic B cells could be delineated by distribution of cell subsets, either by cell frequency or absolute cells number per spleen.

Similarly, flow cytometric analysis of the spleen shows the frequencies of Igκ⁺ and Igλ⁺ B cells in C57BL/6J mice (Figure 4), using a staining panel outlined in Table 4. Average absolute cell number of these frequencies are shown in Table 8. Perturbations in Igκ⁺ and Igλ⁺ Bcells could be delineated by distribution of cell subsets, either by cell frequency or absolute cells number per spleen.

Antibody	Fluorophore	clone
CD19	APC-H7	1D3
B220	APC	RA3-6B2
IgM	PeCy7	II/41
IgD	PerCpCy5.5	11-26c.2a
CD43	FITC	S7
CD23	BUV395	B3B4
CD11b	BV711	M1/70
CD5	BV605	53-7.3

Table 1: Peritoneal B Cell Panel

Antibody	Fluorophore	clone
CD19	APC-H7	1D3
B220	APC	RA3-6B2
IgM	PeCy7	II/41
IgD	PerCpCy5.5	11-26c.2a
CD43	FITC	1B11
CD24 (HSA)	PE	30-F1
C-Kit	BUV395	2B8
BP-1	BV786	BP-1
CD93	BV711	AA4.1
dump channel
CD3	AF700	17-A2
CD11b	AF700	M1/70
GR1 (Ly6C/6G)	AF700	RB6-8C5
Ter119	AF700	TER-119

Table 2: Bone Marrow Maturation Panel

Antibody	Fluorophore	clone
CD19	APC-H7	1D3
B220	APC	RA3-6B2
IgM	PeCy7	II/41
IgD	PerCpCy5.5	11-26c.2a
CD43	FITC	S7
CD23	BUV395	B3B4
CD21/35	BV421	7G6
CD11b	AF700	M1/70
CD5	BV605	53-7.3
CD93	PE	AA4.1

Table 3: Spleen Maturation Panel

Antibody	Fluorophore	clone
CD19	APC-H7	1D3
B220	APC	RA3-6B2
IgM	PeCy7	II/41
IgD	PerCpCy5.5	11-26c.2a
CD3	PB	17-A2
Kappa	FITC	187.1
Lambda	PE	RML-42

Table 4: Spleen Igκ and Igλ Panel

Figure 1: Characterization of B cell populations in the peritoneum. Viable, single cell, size gated peritoneal B cells are first separated from contaminating cells by gating on IgM⁺ cells. B-1 and B-2 cells are then distinguished from each other by absence (B-1) or presence of CD23 (B-2). Next CD5 expression is used to delineated B-1a cells (CD5⁺) from B-1b cells (CD5^-). FMOs were used to empirically determine where to draw gates. Numbers are percentages of each population within the same density plot. Please click here to view a larger version of this figure.

Figure 2: Characterization of B cell subsets in the BM. Viable, single cell, size gated BM B cells are separated from non-B cells by gating on B220⁺ dump^- (where dump refers to CD3/GR-1/CD11b/TER119) cells. CD43 and B220 expression further defines Hardy Fraction A-C' (CD43⁺ B220⁺) and Hardy Fraction D-F (CD43^low/neg B220^+/++). Fraction A-C' is further separated by expression of BP-1 and CD24. Fraction A (BP-1^- CD24^-) corresponds to pre-pro-B cells along with contaminating cells. To separate pre-pro-B cells from contaminating cells in Fraction A, the expression of CD93 and the absence of CD19 are utilized. Fraction B (BP-1^- CD24^int) and Fraction C (BP-1⁺ CD24^int) correspond to early and late pro-B cells, respectively, and Fraction C' (BP-1^+/- CD24⁺) corresponds to early pre-B cells. To separate Fraction D-F, expression of IgM and IgD are utilized. Fraction D corresponds to late pre-B cells (IgM^-/low IgD^-); Fraction E (blue gate, IgM^int/high IgD^-) to both immature (Imm, IgM^int IgD^-) and transitional (Tran, IgM^high IgD^-) B cells; and Fraction F (IgM^int/high IgD⁺) to recirculating mature B cells. FMOs were used to empirically determine where to draw gates. Numbers are percentages of each population within the same density plot. Please click here to view a larger version of this figure.

Figure 3: Characterization of splenic B cell maturation. Viable, single cell, size gated splenic B cells are separated from non-B cells by gating on B220⁺ cells. In order to identify the B-1 subset, CD23^- CD19⁺ cells are identified and defined by expression of CD43. To classify B-2 populations, CD19⁺ cells are separated into transitional (CD93⁺ B220⁺) and mature (CD93^- B220⁺) B cells. Transitional (CD93⁺ B220⁺) cells are further divided into T1 (IgM⁺ CD23^-), T2 (IgM⁺ CD23⁺), and T3 (IgM^int CD23⁺) populations. Mature (CD93^- B220⁺) cells are separated into marginal zone (CD21/35⁺ IgM⁺) and follicular (CD21/35^int IgM^int/+) B cells. The expression of CD23 is further used to separate MZ precursor (CD23⁺ B220⁺) cells from more mature MZ (CD23^- B220⁺) cells. Follicular populations are then delineated into Fol I (IgD⁺ IgM^int) and Fol II (IgD⁺ IgM⁺) cells. FMOs were used to empirically determine where to draw gates. Numbers are percentages of each population within the same density plot Please click here to view a larger version of this figure.

Figure 4: Igκ and Igλ expression of splenic B cells. Viable, single cell, size gated splenic B-cells are separated from non-B-cells by gating on B220⁺ CD3^- cells. B cells are then distinguished by the expression of Igλ and Igκ. Numbers are percentages of each population within the same density plot. Please click here to view a larger version of this figure.

	Absolute Cell Number
Animal Number	Viable peritoneal cells	B cells	B-1a cells	B-1b cells	B-2 cells
1	1.02E+07	4.67E+06	1.28E+06	8.95E+05	2.35E+06
2	9.92E+06	4.52E+06	1.49E+06	9.60E+05	1.91E+06
3	1.15E+07	4.56E+06	1.71E+06	9.19E+05	1.78E+06
Average	1.05E+07	4.58E+06	1.49E+06	9.25E+05	2.01E+06

Table 5: Absolute Cell Numbers of Peritoneal B Cell Subsets

	Absolute Cell Number
Animal Number	Viable bone marrow cells	B cells	Fraction A	Pre-pro	Fraction B	Fraction C	Fraction C'	Fraction D	Immature	Transitional	Fraction F
1	5.05E+07	9.70E+06	1.13E+06	1.95E+05	2.22E+05	9.14E+04	6.31E+05	1.59E+06	4.56E+05	7.81E+05	4.03E+06
2	5.39E+07	1.03E+07	1.14E+06	2.29E+05	2.89E+05	1.22E+05	8.40E+05	2.11E+06	5.39E+05	8.07E+05	3.67E+06
3	5.93E+07	1.01E+07	1.10E+06	2.12E+05	2.84E+05	1.05E+05	9.02E+05	2.72E+06	5.94E+05	7.62E+05	2.59E+06
Average	5.46E+07	1.00E+07	1.12E+06	2.12E+05	2.65E+05	1.06E+05	7.91E+05	2.14E+06	5.29E+05	7.83E+05	3.43E+06

Table 6: Absolute Cell Numbers of Bone Marrow B Cell Subsets

Absolute Cell Number

Animal Number

Viable Spleen Cells

B cells

Transitional B cells

T1 cells

T2 cells

T3 cells

Mature B cells

Follicular I cells

Follicular II cells

Precursor marginal zone cells

Mature marginal zone cells

B-1 cells

1

9.16E+07

4.61E+07

3.66E+06

1.55E+06

1.10E+06

7.16E+05

4.06E+07

2.39E+07

5.27E+06

2.17E+06

3.98E+06

8.83E+05

2

9.97E+07

5.18E+07

4.88E+06

1.97E+06

1.57E+06

1.00E+06

4.49E+07

2.68E+07

7.33E+06

3.42E+06

3.84E+06

8.15E+05

3

1.02E+08

5.34E+07

4.64E+06

1.98E+06

1.41E+06

8.54E+05

4.62E+07

2.81E+07

5.84E+06

3.58E+06

4.02E+06

1.01E+06

Average

9.77E+07

5.04E+07

4.39E+06

1.83E+06

1.36E+06

8.58E+05

4.39E+07

2.63E+07

6.15E+06

3.06E+06

3.94E+06

9.02E+05

Table 7: Absolute Cell Numbers of Splenic B Cell Subsets

	Absolute Cell Number
Animal Number	Viable spleen cells	B cells	Igκ+ B cells	Igλ+ B cells
1	9.16E+07	4.97E+07	4.51E+07	2.46E+06
2	9.97E+07	5.63E+07	5.08E+07	3.16E+06
3	1.02E+08	5.91E+07	5.33E+07	3.24E+06
Average	9.77E+07	5.50E+07	4.97E+07	2.95E+06

Table 8: Absolute Cell Numbers of Igκ and Igλ B Cell Subsets

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 in rare populations. If used to measure complex heterogenous samples, it can detect sub-populations within minutes, on individual cells³³. Sequential gating analysis strategy, most often applied to flow cytometric analysis, can be simple and intuitive when a specific population has to be identified³⁴. Finally, another advantage of flow cytometry is that it is easily adaptable in most academic labs, while under guidance of experienced users. Our protocol successfully describes assessment of B cell populations in the peritoneum, BM, and spleens of mice, by describing and enumerating B-1 populations and delving into the development of B-2 pro-B cells, pre-B cells, immature, transitional, and mature B cells, as well as their surface expression of Igκ or Igλ light chains. Flow cytometry is the most widely used, and easiest method to apply, when investigating B cell development in mice.

While flow cytometry generates invaluable data, there are some limits to this technology when used to investigate the heterogeneity of the immune B cell compartment. Huge data sets can be overwhelming because 10 color staining allows the recognition of more than 1,024 different cell populations³⁴. One must take into consideration that some commonly used lymphoid cell markers have proven to be less specific than originally thought. This can be resolved by employing a multitude of cell surface markers to ascertain gating on desired populations. While flow cytometric analysis can be simple and intuitive, another constraint to flow cytometric analysis is that it typically allows the visualization of only two parameters at a time, though data visualization tools such as t-SNE can be used to cluster cell populations more efficiently when using high parameter flow cytometry. Another important limitation is that the gates used during both the acquisition and analysis are sometimes dependent on the subjectivity of the operator.

For successful adaptation or replication of this protocol, there are several critical parameters that have to be taken into consideration³⁵. Careful consideration must be taken into panel design and fluorochrome selection. It is imperative to pair dim or important antigens with bright fluorochromes. Antibody titration must be carried out to avoid excess antibody binding to cells non-specifically, potentially increasing background staining and decreasing resolution. Antibody titration is carried out by staining a known number of cells with decreasing concentrations of antibodies, to determine the best separation index³⁶. This should be repeated for every lot of antibody. During sample preparation and staining, it is important to assure a single cell suspension by avoiding Ca⁺⁺ and Mg⁺⁺. Additionally, addition of EDTA can help prevent cell aggregation and enzymatic activity which can lead to antibody-mediated stimilulation and internalization of labeled markers. Prior to data acquisition, samples must be properly suspended, filtered and free of aggregates. Spillover of signal from one parameter to another is resolved by using compensation controls, in the form of single stained cells or commercially available compensation beads³⁵. Another important consideration is to have proper controls in each experiment. Unstained cells establish the baseline of autofluorescence. Isotype controls are no longer considered appropriate controls for gating due to non-specific binding. The most important step in helping to make accurate gates is the use of FMO controls. In an FMO control, all conjugated antibodies are present in the stain except the one which is being controlled for. FMO controls enable the measurement of the spread of all the fluorophores into the missing channel and hence allow for setting up gates accordingly. It is critical that enough cells are acquired for added accuracy. As a rule of thumb, at least 2,000 events of the population of interest should be collected. Lastly, compensation controls, whether beads or cells, should be exactly matched to the fluorochromes being utilized and controls must be at least as bright as the experimental samples³⁷.

Overall, low cytometric analysis of B cell compartments is widely used in the immunology field. This technique can be used to investigate perturbations in humoral immunity in both wild type and genetically modified mice, under non-disease states and upon immunological challenge.

Materials

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