D.R.W. is supported by NIH grants AI089972 and AI113217, and by the Mucosal Immunology Studies Team, and holds a Career Award for Medical Scientists from the Burroughs Wellcome Fund.

NOTE: All experiments described here were in accordance with The Institutional Animal Care and Use Committee (IACUC) guidelines and approved by Animal Research Children&#8217;s Hospital (ARCH), Boston, Mass.
1. Preparation of Reagents
<ol>
	<li>1,000x IL-4 Stock: Dilute IL-4 cytokine to 20 &#181;g/ml in H2O or 0.1% bovine serum albumin (BSA). Dispense in 200 &#181;l aliquots to 1.5 ml microcentrifuge tubes and store at -20 &#176;C.</li>
	<li>1,000x anti-CD40: Obtain from the manufacturer at 1.0 mg/ml, store at 4 &#176;C.</li>
	<li>B cell stimulation media: To 425 ml of RPMI-1640 medium, add 75 ml of freshly thawed fetal calf serum (FCS), 10 ml of 1.0 M HEPES buffer, 5 ml L-glutamine stock, 5 ml Penicillin:Streptomycin stock, 5 ml Minimum Essential Medium Non-Essential Amino Acids (MEM-NEAA), and 3.5 &#181;l of 2-mercaptoethanol. Keep media at 4 &#730;C for up to a week. Add IL-4 (20 ng/ml) and anti-CD40 (1.0 &#181;g/ml) immediately before use only to the volume of media required.</li>
	<li>2% FCS-FACS Buffer: Add 10 ml of FCS to 490 ml of 1x PBS. Keep at 4 &#176;C.</li>
	<li>2x trypsin-EDTA solution: Dilute 5 ml of 10x trypsin-EDTA stock solution (5.0 g trypsin, 2.0 g EDTA per liter) in 20 ml of 1x PBS. Store trypsin-EDTA in 2x working aliquots at 4 &#176;C. Allow to warm to room temperature before use.</li>
	<li>Fetal calf serum: Keep thawed FCS at 4 &#730;C for up to two weeks. Place on ice 15 min before the cytoplasmic staining procedure.</li>
	<li>Neutral-buffered formalin: Prepare a 10% formalin solution (3.7% paraformaldehyde). Formalin is toxic and therefore handle it under a laboratory fume hood while wearing appropriate PPE. Store formalin at room temperature in a chemical storage cabinet.</li>
	<li>Methanol: Store absolute methanol (100%) at -20 &#176;C until the cells are ready to be permeabilized. Note that methanol is flammable and should be stored in an appropriate freezer.</li>
</ol>
2. Purification and Activation of B Cells
<ol>
	<li>From a freshly-harvested mouse spleen, prepare a single cell suspension by gently pressing the organ through a 70 &#181;m cell strainer with the plunger of a 3 ml syringe. In a 15 ml conical centrifuge tube, collect cells in 10 ml of 1x PBS and centrifuge at 300 x g for 5 min at 4 &#176;C.</li>
	<li>Decant the supernatant and resuspend pellet in 1 ml of Red Blood Cell Lysing Buffer for 5 min. Neutralize with 13.5 ml of 1x PBS and centrifuge at 300 x g for 5 min.</li>
	<li>(Optional) Magnetically separate the B cells using anti-CD45R(B220)-labeled beads, following the MACS purification protocol according to manufacturer&#8217;s instructions.</li>
	<li>Stimulate purified B cells at 1.0 x 106 cells/ml in warmed B cell stimulation media containing IL-4 (20 ng/ml) and anti-CD40 (1.0 &#181;g/ml) for 5 days at 37 &#176;C. Start with 8 ml of culture divided into 2 wells of a 6-well culture plate (4 ml each).</li>
	<li>Check culture daily to maintain at 1.0 x 106 cells/ml. Adjust the concentration by splitting into other wells and diluting the culture with B cell stimulation media containing fresh IL-4 and anti-CD40.</li>
</ol>
3. Trypsinization, Fixation, and Permeabilization
<ol>
	<li>Trypsinization
	<ol>
		<li>Collect up to 1.0 x 107 of cultured cells in a 15 ml conical tube, centrifuge at 300 x g for 5 min, and decant the supernatant.</li>
		<li>Wash with 10 ml of PBS to remove the residual protein from the cell collection media. Repeat the centrifugation step and decant the supernatant.</li>
		<li>Resuspend the cell pellet in the residual volume after decanting (about 100 &#956;l).</li>
		<li>Carefully add 800 &#956;l of room temperature 0.1% (2x) trypsin-EDTA to the cells. Dispense slowly at a 45&#176; angle down the side of the conical tube.</li>
		<li>Close the tube and mix gently by tilting the tube so the liquid runs along the length of the tube 5-6 times. A stringy, white precipitate may form in the solution. Limit the trypsinization time to under a min.</li>
		<li>Quench the trypsinization reaction with 1 ml of cold FCS. Dilute the FCS immediately by adding 10 ml of cold PBS.</li>
		<li>Centrifuge at 300 x g for 5 min at 4 &#176;C, then decant the supernatant and return the tubes to ice.</li>
	</ol>
	</li>
	<li>Fixation
	<ol>
		<li>Resuspend the cells in the residual volume after decanting. If necessary, bring volume up to 100 &#956;l with cold PBS.</li>
		<li>In a fume hood, add 800 &#956;l of 10% neutral buffered formalin solution to the cells and pipet up and down. The tube will warm upon the addition of formalin. Incubate at 37 &#176;C for 10 min.</li>
	</ol>
	</li>
	<li>Permeabilization
	<ol>
		<li>Set up a benchtop vortex mixer at a gentle shaking speed.</li>
		<li>Draw 9 ml of cold (-20 &#176;C) methanol into a 10 ml serological pipet.</li>
		<li>Hold the tube containing the cells over the vortex mixer and gently start shaking the cells.</li>
		<li>Add cold methanol to the tube so it falls dropwise onto the cells while the tube is still shaking so that the methanol is continuously mixing. After the first 2 ml of methanol the dispensing speed can be increased to a slow stream.</li>
		<li>Let the tube sit on ice for 30 min to complete the permeabilization. Alternatively, the cells can be stored at -20 &#176;C for up to a month.</li>
	</ol>
	</li>
</ol>
4. Fluorescence Staining for IgG1 and IgE Class Switching
<ol>
	<li>Centrifuge sample at 300 x g for 5 min and decant the methanol solution. A white precipitate may pellet with the cells.</li>
	<li>Add 10 ml of room temperature 1x PBS and centrifuge at 300 x g for 5 min to wash cells. Repeat once. Note: Washing the pellet with room temperature PBS dissolves extra precipitate that may form after step 4.1.</li>
	<li>Perform an additional wash with 10 ml of 2% FCS-FACS buffer.</li>
	<li>Distribute samples to a 96-well round bottom plate and centrifuge at 300 x g for 5 min. Decant supernatant and put the plate on ice.</li>
	<li>Stain in 60 &#181;l of 2% FCS-FACS buffer per well with the following antibody dilutions: Anti-IgM RPE/Cy7, 1:200, Anti-IgG1 PE, 1:600, Anti-IgE FITC, 1:100, Anti-CD45R(B220) PerCP-Cy5.5, 1:200</li>
	<li>After 5 min of staining, wash the plate with 150 &#181;l of 2% FCS-FACS buffer and centrifuge for 5 min at 300 x g.</li>
	<li>Resuspend in 400 &#181;l of 2% FCS-FACS buffer and pipet through a 40 &#181;m nylon cell strainer into a 5 ml FACS tube.</li>
</ol>
5. Use the Following Gating Strategy:
<ol>
	<li>Isolate CD45R(B220) positive cells from the blasting lymphocyte population located on the FSC/SSC plot (Figure 1).</li>
	<li>From the B220 positive cells, use the IgE and IgG1 plot to reveal the corresponding populations.</li>
</ol>

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The authors have no disclosures.

Stimulation of mouse splenic B cells in culture with anti-CD40 and IL-4 will simulate T helper type 2 (TH2) interactions, encouraging class switching to IgG1 and IgE5. B cells can be activated for CSR in the context of total splenocytes12 or as purified splenic B cells11. As noted in the protocol (step 2.3), B cell enrichment is optional, and is at the discretion of the experimenter to determine if it would beneficial. Positive magnetic separation of B cells using CD45R(B220)-l...

During immunoglobulin heavy chain (IgH) class switch recombination (CSR) in mice and humans, the IgH &#956; constant region exons (C&#956;) are deleted and replaced by one of several sets of downstream constant region exons (CHs) (e.g. C&#947;, C&#949;, and C&#945;), resulting in a switch from the production of IgM to the production of other Ig classes (e.g. IgG, IgE, or IgA). CSR occurs within switch (S) regions, which are 1-10 kb sequences located 5&#8217; to each set of CHs1. The Activation-Induced Cytidine Deaminase (AID) enzyme initiates CSR via cytidine deamination activity.
IgE is a key mediator of allergic disease2 and a greater understanding of how IgE is produced and regulated may open doors to new therapeutic approaches to atopic disease. In mice and humans, IgE is the most tightly regulated Ig isotype. IgE is normally detected at levels thousands of times less than other IgH isotypes3, but can be highly elevated in disease states4. However, CSR to IgE is incompletely understood. In vitro activation of B cells using IL-4 in combination with either anti-CD40 or LPS, induces CSR to both IgG1 and IgE5. Activated B cells express the low affinity IgE receptor Fc&#949;RII/CD232,6, which binds soluble IgE secreted in culture after CSR. Therefore when analyzing with flow cytometry, B cells with receptor-bound IgE stain similarly to B cells endogenously expressing IgE7. While it is known that mouse B cells express the low affinity IgG receptor Fc&#947;RIIB1 (CD32)8, in our experience it does not appear to interfere with measurement of class switching to IgG1. However, when measuring IgE switching after B cell activation in culture, nonspecific surface-bound IgE may obscure the analysis. With common staining methods, non-IgE-expressing cells stain positively for IgE.
Outlined here is a strategy that has been utilized to conduct CSR assays and detect true IgE-expressing B cells from mice9&#8211;11. Treatment of activated B cells with trypsin, a common lab reagent to digest protein, removes both cytophylic and membrane bound surface IgE. Subsequent permeabilization and staining for cytoplasmic IgE thus reveals the true IgE-producing cells.

<table border="0" cellpadding="0" cellspacing="0" width="958">
	<tbody>
		<tr height="37">
			<td height="37" style="height:37px;width:301px;">Name</td>
			<td style="width:179px;">Company</td>
			<td style="width:135px;">Catalog Number</td>
			<td style="width:343px;">Comments</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Anti-Mouse IgE FITC</td>
			<td>BD Pharmingen</td>
			<td>553415</td>
			<td>clone R35-72</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Rat Anti-Mouse IgG1 PE</td>
			<td>BD Pharmingen</td>
			<td>550083</td>
			<td>clone A85-1</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Methanol</td>
			<td>BDH</td>
			<td>BDH1135-4LP</td>
			<td>Keep at -20 &#176;C</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Falcon Cell Strainer 40 &#181;m Nylon</td>
			<td>Corning</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Falcon Cell Strainer 70 &#181;m Nylon</td>
			<td>Corning</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Anti-Hu/Mo CD45R(B220) PerCP-Cy5.5</td>
			<td>eBioscience</td>
			<td>45-4052-82</td>
			<td>clone RA3-6B2</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">anti-Mouse/Rat CD40</td>
			<td>eBioscience</td>
			<td>16-0402-85</td>
			<td>clone HM40-3</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">RPMI Medium 1640</td>
			<td>Gibco</td>
			<td>11875-093</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">HEPES (1M)</td>
			<td>Gibco</td>
			<td>15630-080</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">MEM-NEAA (100X)</td>
			<td>Gibco</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Phosphate Buffered Saline (10X)</td>
			<td>Lifetechnologies (Corning)</td>
			<td>46-013-CM</td>
			<td></td>
		</tr>
		<tr height="66">
			<td height="66" style="height:67px;">MACS CD45R (B220) microbeads&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>130-049-501</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">MACS purification column</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">IL-4</td>
			<td>PeproTech</td>
			<td>214-14</td>
			<td>Reconstitute in water or 0.1% BSA</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Formalin Solution, Neutral Buffered, 10%</td>
			<td>Sigma Aldrich</td>
			<td>HT501128-4L</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Trypsin-EDTA Solution (10X)</td>
			<td>Sigma Aldrich</td>
			<td>T4174-100mL</td>
			<td>5.0g Trypsin, 2.0g EDTA&#376;4Na per Liter of 0.9% NaCl</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Penicillin-Streptomycin</td>
			<td>Sigma Aldrich</td>
			<td>P0781-100mL</td>
			<td>10,000U Penicillin; 10 mg/mL Streptomycin (100X)</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">L-Glutamine 200mM</td>
			<td>Sigma Aldrich</td>
			<td>G7513</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">2-mercaptoethanol (99%)</td>
			<td>Sigma Aldrich</td>
			<td>M6250-100mL</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Red cell lysis buffer</td>
			<td>Sigma Aldrich</td>
			<td>R7757</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Rat Anti-Mouse IgM RPE/cy7</td>
			<td>SouthernBiotech</td>
			<td>1140-17</td>
			<td>clone 1B4B1</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">HyClone Fetal Calf Serum</td>
			<td>Thermo</td>
			<td>SH30910.03</td>
			<td></td>
		</tr>
		<tr height="161">
			<td height="161" style="height:161px;">B cell Stimulation media</td>
			<td></td>
			<td></td>
			<td style="width:343px;">B cell stimulation media consists of RPMI with fetal calf serum (15%), 20 mM HEPES, 1X MEM-NEAA, 2.0mM L-glutamine,&#160; 1X Penicillin-Streptomycin (Penicillin:100 U/ml, Streptomycin: 100 &#181;g/ml), Beta-mercaptoethanol (7 &#181;L/L), IL-4 (20 ng/mL), and anti-CD40 (1 &#181;g/mL)</td>
		</tr>
	</tbody>
</table>

detection of true ige-expressing mouse b lineage cells

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH isotypes, such as IgA, IgE and IgG. Measurement of IgH CSR in vitro is a key method for the study of a number of biologic processes ranging from DNA recombination and repair to aspects of molecular and cellular immunology. In vitro CSR assay involves the flow cytometric measurement surface Ig expression on activated B cells. While measurement of IgA and IgG subclasses is straightforward, measurement of IgE by this method is problematic due to soluble IgE binding to Fc&#949;RII/CD23 expressed on the surface of activated B cells. Here we describe a unique procedure for accurate measurement of IgE-producing mouse B cells that have undergone CSR in culture. The method is based on trypsin-mediated cleavage of IgE-CD23 complexes on cell surfaces, allowing for detection of IgE-producing B lineage cells by cytoplasmic staining. This procedure offers a convenient solution for flow cytometric analysis of CSR to IgE.

This procedure has been successfully implemented to study CSR to IgE in mouse B cells. To demonstrate the efficiency in measuring CSR, we stimulated mouse splenic B cells with anti-CD40 and IL-4 as described previously11. After five days of stimulation, cells were collected and processed using the protocol described above and stained with fluorescently-labeled IgM, IgG1, IgE, and B220 (CD45R) antibodies. Gated on the blasting lymphocytes (Figure 1), a clear population of IgE+ cells cannot be c...

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Detection of True IgE-expressing Mouse B Lineage Cells

In vitro analysis of class switch recombination in mice is challenging due to cytophilic IgE molecules bound to Fc receptors on the surface of B cells. We describe a method for IgE detection using trypsin-mediated cleavage of surface-bound IgE prior to fixation and permeabilization for cytoplasmic fluorescence staining.

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH ...

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Immunology and Infection

10.8K Views.  Brigham and Women's Hospital and Harvard Medical School. In vitro analysis of class switch recombination in mice is challenging due to cytophilic IgE molecules bound to Fc receptors on the surface of B cells. We describe a method for IgE detection using trypsin-mediated cleavage of surface-bound IgE prior to fixation and permeabilization for cytoplasmic fluorescence staining.

Video: Detection of True IgE-expressing Mouse B Lineage Cells

Murine Model of CD40-activation of B cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients1-4. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro5. Furthermore they efficiently take up, process and present antigens to T cells6,7. CD40-B cells were shown to not only prime na&iacute;ve, but also expand memory T cells8,9. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa)11. Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (&gt;90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach12.

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

Induction and Assessment of Class Switch Recombination in Purified Murine B Cells

Humoral immunity is the branch of the immune system maintained by B cells and mediated through the secretion of antibodies. Upon B cell activation, the immunoglobulin locus undergoes a series of genetic modifications to alter the binding capacity and effector function of secreted antibodies. This process is highlighted by a genomic recombination event known as class switch recombination (CSR) in which the default IgM antibody isotype is substituted for one of IgG, IgA, or IgE. Each isotype possesses distinct effector functions thereby making CSR crucial to the maintenance of immunity. Diversification of the immunoglobulin locus is mediated by the enzyme activation-induced cytidine deaminase (AID). A schematic video describing this process in detail is available online (http://video.med.utoronto.ca/videoprojects/immunology/aam.html). AID's activity and the CSR pathway are commonly studied in the assessment of B cell function and humoral immunity in mice. The protocol outlined in this report presents a method of B cell isolation from murine spleens and subsequent stimulation with bacterial lipopolysaccharide (LPS) to induce class switching to IgG3 (for other antibody isotypes see Table 1). In addition, the fluorescent cell staining dye Carboxyfluorescein succinimidyl ester (CFSE) is used to monitor cell division of stimulated cells, a process crucial to isotype switching 1, 2. The regulation of AID and the mechanism by which CSR occurs are still unclear and thus in vitro class switch assays provide a reliable method for testing these processes in various mouse models. These assays have been previously used in the context of gene deficiency using knockout mice 3. Furthermore, in vitro switching of B cells can be preceded by viral transduction to modulate gene expression by RNA knockdown or transgene expression 4-6. The data from these types of experiments have impacted our understanding of AID activity, resolution of the CSR reaction, and antibody-mediated immunity in the mouse.

Following antigen exposure, subpopulations of activated B cells undergo a process known as class switch recombination (CSR) to produce antibody isotypes with distinct effector functions. The protocol outlined in this report explains how CSR can be induced and analyzed in vitro for the purposes of studying B cell function.

Humoral immunity is the branch of the immune system maintained by B cells and mediated through the secretion of antibodies. Upon B cell activation, the ...

Recombinant Retroviral Production and Infection of B Cells

The transgenic expression of genes in eukaryotic cells is a powerful reverse genetic approach in which a gene of interest is expressed under the control of a heterologous expression system to facilitate the analysis of the resulting phenotype. This approach can be used to express a gene that is not normally found in the organism, to express a mutant form of a gene product, or to over-express a dominant-negative form of the gene product. It is particularly useful in the study of the hematopoetic system, where transcriptional regulation is a major control mechanism in the development and differentiation of B cells 1, reviewed in 2-4.
Mouse genetics is a powerful tool for the study of human genes and diseases. A comparative analysis of the mouse and human genome reveals conservation of synteny in over 90% of the genome 5. Also, much of the technology used in mouse models is applicable to the study of human genes, for example, gene disruptions and allelic replacement 6. However, the creation of a transgenic mouse requires a great deal of resources of both a financial and technical nature. Several projects have begun to compile libraries of knock out mouse strains (KOMP, EUCOMM, NorCOMM) or mutagenesis induced strains (RIKEN), which require large-scale efforts and collaboration 7. Therefore, it is desirable to first study the phenotype of a desired gene in a cell culture model of primary cells before progressing to a mouse model. 
Retroviral DNA integrates into the host DNA, preferably within or near transcription units or CpG islands, resulting in stable and heritable expression of the packaged gene of interest while avoiding transcriptional silencing 8 9. The genes are then transcribed under the control of a high efficiency retroviral promoter, resulting in a high efficiency of transcription and protein production. Therefore, retroviral expression can be used with cells that are difficult to transfect, provided the cells are in an active state during mitosis. Because the structural genes of the virus are contained within the packaging cell line, the expression vectors used to clone the gene of interest contain no structural genes of the virus, which both eliminates the possibility of viral revertants and increases the safety of working with viral supernatants as no infectious virions are produced 10. 
Here we present a protocol for recombinant retroviral production and subsequent infection of splenic B cells. After isolation, the cultured splenic cells are stimulated with Th derived lymphokines and anti-CD40, which induces a burst of B cell proliferation and differentiation 11. This protocol is ideal for the study of events occurring late in B cell development and differentiation, as B cells are isolated from the spleen following initial hematopoetic events but prior to antigenic stimulation to induce plasmacytic differentiation.

An efficient system of structure and function analysis of a gene in an ex vivo culture of splenic B-lymphocytes is described. This method takes advantage of recombinant retroviral production in a helper free, ecotrophic packaging cell line. Stable, heritable expression of a gene of interest within primary lymphocytes is achieved leading to generation of surface antibodies on B cells undergoing class switch recombination.

The transgenic expression of genes in eukaryotic cells is a powerful reverse genetic approach in which a gene of interest is expressed under the control ...

Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency and type of chromosome aberrations in different cell types allows defects in DNA repair pathways to be elucidated. Understanding mammalian DNA repair biology has been greatly helped by the production of mice with knockouts in specific genes. The goal of this protocol is to quantify genomic instability in mouse B lymphocytes. Labeling of the telomeres using PNA-FISH probes (peptide nucleic acid - fluorescent in situ hybridization) facilitates the rapid analysis of genomic instability in metaphase chromosome spreads. B cells have specific advantages relative to fibroblasts, because they have normal ploidy and a higher mitotic index. Short-term culture of B cells therefore enables precise measurement of genomic instability in a primary cell population which is likely to have fewer secondary genetic mutations than what is typically found in transformed fibroblasts or patient cell lines.

DNA repair pathways are essential for maintenance of genomic integrity and preventing mutation and cancer. The goal of this protocol is to quantify genomic instability by direct observation of chromosome aberrations in metaphase spreads from mouse B cells using fluorescent in situ hybridization (FISH) for telomeric DNA repeats.

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency ...

Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, including the development of immunotherapeutics. However, the techniques to identify and produce such antibodies tend to be arduous and sometimes the heavy and light chain pair of the antibodies are dissociated. Here, we describe a relatively simple, straightforward protocol to produce human recombinant monoclonal antibodies from human peripheral blood mononuclear cells using immortalization with Epstein-Barr Virus (EBV) and Toll-like receptor 9 activation. With an adequate staining, B cells producing antibodies can be isolated for subsequent immortalization and clonal expansion. The antibody transcripts produced by the immortalized B cell clones can be amplified by PCR, sequenced as corresponding heavy and light chain pairs and cloned into immunoglobulin expression vectors. The antibodies obtained with this technique can be powerful tools to study relevant human immune responses, including autoimmunity, and create the basis for new therapeutics.

Synthesis of human monoclonal antibodies is the first step in studies aimed at unraveling the pathophysiological mechanisms of auto-antibody-mediated immune responses. We have developed a protocol to generate recombinant human immunoglobulin G (IgG) monoclonal antibodies from blood sorted B cells, including B-cell isolation, antibody cloning and in vitro synthesis.

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, ...

Rapid Molecular Detection and Differentiation of Influenza Viruses A and B

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality every year. The Influenza A and B assay was the first CLIA-waived molecular rapid flu test available. The Influenza A and B test works by employing isothermal amplification with influenza-specific primers followed by target detection with molecular beacon probes. Here, the performance of the Influenza A and B assay on frozen, archived nasopharyngeal swab (NPS) specimens stored in viral transport medium (VTM) were compared to a respiratory panel assay.
The performance of the Influenza A and B assay was evaluated by comparing the results to the respiratory panel reference method. The sensitivity for total influenza virus A was 67.5% (95% CI (CI), 56.6-78.5) and the specificity was 86.9% (CI, 71.0-100). For influenza virus B testing, the sensitivity and specificity were 90.2% (CI, 68.5-100) and 98.8% (CI, 68.5-100), respectively.
This system has the advantage of a significantly shorter test time than any other currently available molecular assay and the simple, pipette-free procedure runs on a fully integrated, closed, small-footprint system. Overall, the Influenza A and B assay evaluated in this study has the potential to serve as a point-of-care rapid influenza diagnostic test.

We describe a rapid, molecular-based Influenza A and B assay. The Influenza assay detects each target within 15 min by employing isothermal amplification with influenza-specific primers followed by target detection with molecular beacon probes. The Influenza A and B assay is user-friendly and required minimal hands-on time to perform.

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality ...

Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate and enrich these rare cells for studies of their phenotype and biology. Approaches are inevitably based on the principle that B cells recognize native antigen by virtue of cell surface receptors that are representative in specificity of antibodies that will eventually be secreted by their differentiated daughters. Perhaps the most obvious approach to the problem involves use of fluorochrome-conjugated antigens in conjunction with fluorescence-activated cell sorting (FACS). However, the utility of these methods is limited by cell frequency and the achievable rate of analysis and isolation by electronic sorting. A novel method to enrich rare antigen-specific B cells using magnetic nanoparticles that results in high yield enrichment of antigen-reactive B cells from large starting cell populations is described. This method enables improved monitoring of the phenotype and biology of antigen reactive cells before and following in vivo antigen encounter, such as after immunization or during development of autoimmunity.

A simple yet effective method that employs magnetic nanoparticles to detect and enrich antigen-reactive B cells for functional and phenotypic analysis is described.

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate ...

Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote inflammatory and autoinflammatory diseases. Inflammasomes are activated by cytosolic pattern recognition receptors, including members of the NOD-like receptor (NLR) family. These receptors oligomerize upon the detection of microbial or damage-associated stimuli. Subsequent recruitment of the adaptor protein ASC forms a microscopically visible inflammasome complex, which activates caspase-1 through proximity-induced auto-activation. Following the activation, caspase-1 cleaves pro-IL-1&#946; and pro-IL-18, leading to the activation and secretion of these pro-inflammatory cytokines. Caspase-1 also mediates the inflammatory form of cell death termed pyroptosis, which features the loss of membrane integrity and cell lysis. Caspase-1 cleaves gasdermin D, releasing the N-terminal fragment which forms plasma membrane pores, leading to osmotic lysis.
In vitro, the activation of caspase-1 can be determined by labeling bone marrow-derived macrophages with the caspase-1 activity probe FAM-YVAD-FMK and by labeling the cells with antibodies against the adaptor protein ASC. This technique allows the identification of inflammasome formation and caspase-1 activation in individual cells using fluorescence microscopy. Pyroptotic cell death can be detected by measuring the release of cytosolic lactate dehydrogenase into the medium. This procedure is simple, cost effective and performed in a 96-well plate format, allowing adaptation for screening. In this manuscript, we show that activation of the NLRP3 inflammasome by nigericin leads to the co-localization of the adaptor protein ASC and active caspase-1, leading to pyroptosis.

We describe the detection of NLRP3 inflammasome activation on cellular basis using fluorescence microscopy and staining for active caspase-1 and the adaptor, ASC. A lactate dehydrogenase release assay is presented to detect pyroptotic lysis on a population basis. These techniques can be adapted to study many aspects of inflammasome biology.

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote ...

Identification and Isolation of Oligopotent and Lineage-committed Myeloid Progenitors from Mouse Bone Marrow

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for hematological and immunological analyses. For example, studies of the cellular and molecular properties of myeloid progenitor populations can reveal mechanisms underlying leukemic transformation, or demonstrate how the immune system responds to pathogen exposure. Previously described flow cytometry strategies for myeloid progenitor identification have enabled significant advances in many fields, but the fractions they identify are very heterogeneous. The most commonly used gating strategies define bone marrow fractions that are enriched for the desired populations, but also contain large numbers of &#34;contaminating&#34; progenitors. Our recent studies have resolved much of this heterogeneity, and the protocol we present here permits the isolation of 6 subpopulations of oligopotent and lineage-committed myeloid progenitors from 2 previously described bone marrow fractions. The protocol describes 3 stages: 1) isolation of bone marrow cells, 2) enrichment for hematopoietic progenitors by magnetic-activated cell sorting (lineage depletion by MACS), and 3) identification of myeloid progenitor subsets by flow cytometry (including fluorescence-activated cell sorting, FACS, if desired). This approach permits progenitor quantification and isolation for a variety of in vitro and in vivo applications, and has already yielded novel insight into pathways and mechanisms of neutrophil, monocyte, and DC differentiation.

We demonstrate how to identify and isolate 6 subsets of myeloid progenitors from murine bone marrow using a combination of magnetic and fluorescence sorting (MACS and FACS). This protocol can be used for in vitro culture assays (methylcellulose or liquid cultures), in vivo adoptive transfer experiments, and RNA/protein analyses.

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for ...

Detection of Low Copy Number Integrated Viral DNA Formed by In Vitro Hepatitis B Infection

Hepatitis B Virus (HBV) is a common blood-borne pathogen causing liver cancer and liver cirrhosis resulting from chronic infection. The virus generally replicates through an episomal DNA intermediate; however, integration of HBV DNA fragments into the host genome has been observed, even though this form is not necessary for viral replication. The exact purpose, timing, and mechanism(s) by which HBV DNA integration occurs is not yet clear, but recent data show that it occurs very early after infection. Here, the in vitro generation and detection of HBV DNA integrations are described in detail. Our protocol specifically amplifies single copies of virus-cell DNA integrations and allows both absolute quantification and single-base pair resolution of the junction sequence. This technique has been applied to various HBV-susceptible cell types (including primary human hepatocytes), to various HBV mutants, and in conjunction with various drug exposures. We foresee this technique becoming a key assay to determine the underlying molecular mechanisms of this clinically relevant phenomenon.

Detection of Low Copy Number Integrated Viral DNA Formed by In Vitro Hepatitis B Infection

We describe here the in vitro generation of HBV DNA via a Hepatitis B virus infection system and the highly sensitive detection of its (1&#8211;2 copies) integration using inverse nested PCR.

Hepatitis B Virus (HBV) is a common blood-borne pathogen causing liver cancer and liver cirrhosis resulting from chronic infection. The virus generally ...