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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<li>Gould, H. J., Sutton, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((IgE+in+allergy+and+asthma+today%5BTitle%5D)+AND+%22Nature+reviews.+Immunology%22%5BJournal%5D)">IgE in allergy and asthma today.</a> Nature reviews. Immunology. 8, 205-217 (2008).</li>
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<li>Boboila, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Alternative+end-joining+catalyzes+class+switch+recombination+in+the+absence+of+both+Ku70+and+DNA+ligase+4%5BTitle%5D)+AND+%22The+Journal+of+experimental+medicine%22%5BJournal%5D)">Alternative end-joining catalyzes class switch recombination in the absence of both Ku70 and DNA ligase 4.</a> The Journal of experimental medicine. 207, 417-427 (2010).</li>
<li>Nimmerjahn, F., Ravetch, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fcgamma+receptors+as+regulators+of+immune+responses%5BTitle%5D)+AND+%22Nature+reviews.+Immunology%22%5BJournal%5D)">Fcgamma receptors as regulators of immune responses.</a> Nature reviews. Immunology. 8 (1), 34-47 (2008).</li>
<li>Callen, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((53BP1+mediates+productive+and+mutagenic+DNA+repair+through+distinct+phosphoprotein+interactions%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">53BP1 mediates productive and mutagenic DNA repair through distinct phosphoprotein interactions.</a> Cell. 153 (6), 153-1280 (2013).</li>
<li>Wesemann, D. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reprogramming+IgH+isotype-switched+B+cells+to+functional-grade+induced+pluripotent+stem+cells%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Reprogramming IgH isotype-switched B cells to functional-grade induced pluripotent stem cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 109 (34), 13745-13750 (2012).</li>
<li>Wesemann, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Immature+B+cells+preferentially+switch+to+IgE+with+increased+direct+S%C2%B5+to+S%CE%B5+recombination%5BTitle%5D)+AND+%22The+Journal+of+experimental+medicine%22%5BJournal%5D)">Immature B cells preferentially switch to IgE with increased direct Sµ to Sε recombination.</a> The Journal of experimental medicine. 208 (13), 2733-2746 (2011).</li>
<li>Zarrin, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+evolutionarily+conserved+target+motif+for+immunoglobulin+class-switch+recombination%5BTitle%5D)+AND+%22Nature+immunology%22%5BJournal%5D)">An evolutionarily conserved target motif for immunoglobulin class-switch recombination.</a> Nature immunology. 5, 1275-1281 (2004).</li>
<li>Fan, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+a+class-specific+polyclonal+antibody-based+indirect+competitive+ELISA+for+detecting+fluoroquinolone+residues+in+milk%5BTitle%5D)+AND+%22Journal+of+Zhejiang+University.+Science.+B%22%5BJournal%5D)">Development of a class-specific polyclonal antibody-based indirect competitive ELISA for detecting fluoroquinolone residues in milk.</a> Journal of Zhejiang University. Science. B. 13 (7), 545-554 (2012).</li>
<li>Perez, O., Krutzik, P., Nolan, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Flow+cytometric+analysis+of+kinase+signaling+cascades%5BTitle%5D)+AND+%22Methods+in+molecular+biology+%28Clifton%2C+N.J.%29%22%5BJournal%5D)">Flow cytometric analysis of kinase signaling cascades.</a> Methods in molecular biology (Clifton, N.J.). 263, Clifton, NJ. 67-94 (2004).</li>
<li>Jamur, M., Oliver, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cell+fixatives+for+immunostaining%5BTitle%5D)+AND+%22Methods+in+molecular+biology+%28Clifton%2C+N.J.%29%22%5BJournal%5D)">Cell fixatives for immunostaining.</a> Methods in molecular biology (Clifton, N.J.). 588, 55-61 (2010).</li>
<li>Ishizaka, T., Ishizaka, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanisms+of+passive+sensitization.+IV.+Dissociation+of+IgE+molecules+from+basophil+receptors+at+acid+pH%5BTitle%5D)+AND+%22Journal+of+immunology+%28Baltimore%2C+MD.+%3A+1950%29%22%5BJournal%5D)">Mechanisms of passive sensitization. IV. Dissociation of IgE molecules from basophil receptors at acid pH.</a> Journal of immunology (Baltimore, MD. : 1950). 112 (3), 1078-1084 (1950).</li>
<li>Erazo, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Unique+Maturation+Program+of+the+IgE+Response+In+Vivo%5BTitle%5D)+AND+%22Immunity%22%5BJournal%5D)">Unique Maturation Program of the IgE Response In Vivo.</a> Immunity. 26, 191-203 (2007).</li>
<li>Xiong, H., Dolpady, J., Wabl, M., Curotto de Lafaille, M. A., Lafaille, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sequential+class+switching+is+required+for+the+generation+of+high+affinity+IgE+antibodies%5BTitle%5D)+AND+%22Journal+of+Experimental+Medicine%22%5BJournal%5D)">Sequential class switching is required for the generation of high affinity IgE antibodies.</a> Journal of Experimental Medicine. 209, 353-364 (2012).</li>
<li>Yang, Z., Sullivan, B., Allen, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fluorescent+in+vivo+detection+reveals+that+IgE%28%2B%29+B+cells+are+restrained+by+an+intrinsic+cell+fate+predisposition%5BTitle%5D)+AND+%22Immunity%22%5BJournal%5D)">Fluorescent in vivo detection reveals that IgE(+) B cells are restrained by an intrinsic cell fate predisposition.</a> Immunity. 36 (5), 857-872 (2012).</li>
<li>Berkowska, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+IgE%28%2B%29+B+cells+are+derived+from+T+cell-dependent+and+T+cell-independent+pathways%5BTitle%5D)+AND+%22The+Journal+of+allergy+and+clinical+immunology%22%5BJournal%5D)">Human IgE(+) B cells are derived from T cell-dependent and T cell-independent pathways.</a> The Journal of allergy and clinical immunology. , (2014).</li>
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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><tbody><tr><td>Anti-Mouse IgE FITC</td><td>BD Pharmingen</td><td>553415</td><td>clone R35-72</td></tr><tr><td>Rat Anti-Mouse IgG1 PE</td><td>BD Pharmingen</td><td>550083</td><td>clone A85-1</td></tr><tr><td>Methanol</td><td>BDH</td><td>BDH1135-4LP</td><td>Keep at -20 &amp;deg;C</td></tr><tr><td>Falcon Cell Strainer 40 &amp;micro;m Nylon</td><td>Corning</td><td>352340</td><td></td></tr><tr><td>Falcon Cell Strainer 70 &amp;micro;m Nylon</td><td>Corning</td><td>352350</td><td></td></tr><tr><td>Anti-Hu/Mo CD45R(B220) PerCP-Cy5.5</td><td>eBioscience</td><td>45-4052-82</td><td>clone RA3-6B2</td></tr><tr><td>anti-Mouse/Rat CD40</td><td>eBioscience</td><td>16-0402-85</td><td>clone HM40-3</td></tr><tr><td>RPMI Medium 1640</td><td>Gibco</td><td>11875-093</td><td></td></tr><tr><td>HEPES (1M)</td><td>Gibco</td><td>15630-080</td><td></td></tr><tr><td>MEM-NEAA (100x)</td><td>Gibco</td><td>11140-050</td><td></td></tr><tr><td>Phosphate Buffered Saline (10x)</td><td>Lifetechnologies (Corning)</td><td>46-013-CM</td><td></td></tr><tr><td>MACS CD45R (B220) microbeads&amp;nbsp;</td><td>Miltenyi Biotec</td><td>130-049-501</td><td></td></tr><tr><td>MACS purification column</td><td>Miltenyi Biotec</td><td>130-042-401</td><td></td></tr><tr><td>IL-4</td><td>PeproTech</td><td>214-14</td><td>Reconstitute in water or 0.1% BSA</td></tr><tr><td>Formalin Solution, Neutral Buffered, 10%</td><td>Sigma Aldrich</td><td>HT501128-4L</td><td></td></tr><tr><td>Trypsin-EDTA Solution (10x)</td><td>Sigma Aldrich</td><td>T4174-100 ml</td><td>5.0 g Trypsin, 2.0 g EDTA&amp;#8226;4 Na per Liter of 0.9% NaCl</td></tr><tr><td>Penicillin-Streptomycin</td><td>Sigma Aldrich</td><td>P0781-100 ml</td><td>10,000 U Penicillin; 10 mg/ml Streptomycin (100x)</td></tr><tr><td>L-Glutamine 200 mM</td><td>Sigma Aldrich</td><td>G7513</td><td></td></tr><tr><td>2-mercaptoethanol (99%)</td><td>Sigma Aldrich</td><td>M6250-100 ml</td><td></td></tr><tr><td>Red cell lysis buffer</td><td>Sigma Aldrich</td><td>R7757</td><td></td></tr><tr><td>Rat Anti-Mouse IgM RPE/Cy7</td><td>SouthernBiotech</td><td>1140-17</td><td>clone 1B4B1</td></tr><tr><td>HyClone Fetal Calf Serum</td><td>Thermo</td><td>SH30910.03</td><td></td></tr><tr><td>B cell Stimulation media</td><td></td><td></td><td>B cell stimulation media consists of RPMI with fetal calf serum (15%), 20 mM HEPES, 1x MEM-NEAA, 2.0 mM L-glutamine,&amp;nbsp; 1x Penicillin-Streptomycin (Penicillin: 100 U/ml, Streptomycin: 100 &amp;micro;g/ml), Beta-mercaptoethanol (7 &amp;micro;L/L), IL-4 (20 ng/ml), and anti-CD40 (1 &amp;micro;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...

Watch this Scientific Journal Video about Detection of True IgE-expressing Mouse B Lineage Cells at JoVE.com

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 ...

detection-of-true-ige-expressing-mouse-b-lineage-cells

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Immunology and Infection

11.0K 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

Detection and Isolation of Viable Mouse IL-17-Secreting T Cells

The MACS Cytokine Secretion Assay technology allows detection of secreted cytokines on the single cell level and sensitive isolation of viable cytokine-secreting cells. In order to label IL-17-secreting cells, a single cell suspension of mouse splenocytes is prepared and stimulated at 37&deg;C with PMA/ionomycin to induce cytokine secretion. To stop secretion cells are then placed on ice and are exposed to the IL-17 Catch Reagent   a bi-specific antibody that binds to CD45 on the cell surface of leukocytes and to IL-17 as it is secreted and caught near the cell surface. Secretion is then re-started by increasing the temperature to 37&deg;C and IL-17 is trapped by the Catch Reagent. Secretion is then stopped again, by placing cells on ice. To detect the trapped IL-17, cells are incubated with a second IL-17-specific antibody conjugated to biotin and an Anti-Biotin-PE antibody. Cells can now be directly analyzed by flow cytometry or prepared for isolation and enrichment by subsequent labeling with Anti-PE conjugated MicroBeads.

This procedure describes the detection and isolation of mouse TH17 leukocytes that actively secrete IL-17 upon stimulation.

The MACS Cytokine Secretion Assay technology allows detection of secreted cytokines on the single cell level and sensitive isolation of viable ...

Biology

Visualization of IL-22-expressing Lymphocytes Using Reporter Mice

Reporter mice have been widely used to observe the localization of expression of targeted genes. This protocol focuses on a strategy to establish a new transgenic reporter mouse model. We chose to visualize interleukin (IL) 22 gene expression because this cytokine has important activities in the intestine, where it contributes to repair tissues damaged by inflammation. Reporter systems offer considerable advantages over other methods of identifying products in vivo. In the case of IL-22, other studies had first isolated cells from tissues and then re-stimulated the cells in vitro. IL-22, which is normally secreted, was trapped inside cells using a drug, and intracellular staining was used to visualize it. This method identifies cells capable of producing IL-22, but it does not determine whether they were doing so in vivo. The reporter design includes inserting a gene for a fluorescent protein (tdTomato) into the IL-22 gene in such a way that the fluorescent protein cannot be secreted and therefore remains trapped inside the producing cells in vivo. Fluorescent producers can then be visualized in tissue sections or by ex vivo analysis through flow cytometry. The actual construction process for the reporter included recombineering a bacterial artificial chromosome that contained the IL-22 gene. This engineered chromosome was then introduced into the mouse genome. Homeostatic IL-22 reporter expression was observed in different mouse tissues, including the spleen, thymus, lymph nodes, Peyer&#39;s patch, and intestine, by flow cytometry analysis. Colitis was induced by T-cell (CD4+CD45RBhigh) transfer, and reporter expression was visualized. Positive T cells were first present in the mesenteric lymph nodes, and then they accumulated inside the lamina propria of the distal small intestine and colon tissues. The strategy using BACs gave good-fidelity reporter expression compared to IL-22 expression, and it is simpler than knock-in procedures.

We describe here a transgenic reporter mouse model to visualize the IL-22-producing cells inside different mouse tissues. This method can be used to track the location of other cytokines or secretary proteins in the mouse.

Reporter mice have been widely used to observe the localization of expression of targeted genes. This protocol focuses on a strategy to establish a new ...

Identification and Analysis of Mouse Erythroid Progenitors using the CD71/TER119 Flow-cytometric Assay

The study of erythropoiesis aims to understand how red cells are formed from earlier hematopoietic and erythroid progenitors. Specifically, the rate of red cell formation is regulated by the hormone erythropoietin (Epo), whose synthesis is triggered by tissue hypoxia. A threat to adequate tissue oxygenation results in a rapid increase in Epo, driving an increase in erythropoietic rate, a process known as the erythropoietic stress response. The resulting increase in the number of circulating red cells improves tissue oxygen delivery. An efficient erythropoietic stress response is therefore critical to the survival and recovery from physiological and pathological conditions such as high altitude, anemia, hemorrhage, chemotherapy or stem cell transplantation. 
The mouse is a key model for the study of erythropoiesis and its stress response. Mouse definitive (adult-type) erythropoiesis takes place in the fetal liver between embryonic days 12.5 and 15.5, in the neonatal spleen, and in adult spleen and bone marrow. Classical methods of identifying erythroid progenitors in tissue rely on the ability of these cells to give rise to red cell colonies when plated in Epo-containing semi-solid media. Their erythroid precursor progeny are identified based on morphological criteria. Neither of these classical methods allow access to large numbers of differentiation-stage-specific erythroid cells for molecular study. Here we present a flow-cytometric method of identifying and studying differentiation-stage-specific erythroid progenitors and precursors, directly in the context of freshly isolated mouse tissue. The assay relies on the cell-surface markers CD71, Ter119, and on the flow-cytometric 'forward-scatter' parameter, which is a function of cell size. The CD71/Ter119 assay can be used to study erythroid progenitors during their response to erythropoietic stress in vivo, for example, in anemic mice or mice housed in low oxygen conditions. It may also be used to study erythroid progenitors directly in the tissues of genetically modified adult mice or embryos, in order to assess the specific role of the modified molecular pathway in erythropoiesis.

A flow-cytometric method for identification and molecular analysis of differentiation-stage-specific murine erythroid progenitors and precursors, directly in freshly &#x2013;harvested mouse bone marrow, spleen or fetal liver. The assay relies on cell-surface markers CD71, Ter119, and cell size.

The study of erythropoiesis aims to understand how red cells are formed from earlier hematopoietic and erythroid progenitors. Specifically, the rate of ...

Characterization of Thymus-dependent and Thymus-independent Immunoglobulin Isotype Responses in Mice Using Enzyme-linked Immunosorbent Assay

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a formidable defense against invading pathogens via diverse mechanisms. One major goal of vaccination is to induce protective antigen-specific antibodies to prevent life-threatening infections. Both thymus-dependent (TD) and thymus-independent (TI) antigens can elicit robust antigen-specific IgM responses and can also induce the production of isotype-switched antibodies (IgG, IgA and IgE) as well as the generation of memory B cells with the help provided by antigen presenting cells (APCs). Here, we describe a protocol to characterize TD and TI Ig isotype responses in mice using enzyme-linked immunosorbent assay (ELISA). In this protocol, TD and TI Ig responses are elicited in mice by intraperitoneal (i.p.) immunization with hapten-conjugated model antigens TNP-KLH (in alum) and TNP-polysaccharide (in PBS), respectively. To induce TD memory response, a booster immunization of TNP-KLH in alum is given at 3 weeks after the first immunization with the same antigen/adjuvant. Mouse sera are harvested at different time points before and after immunization. Total serum Ig levels and TNP-specific antibodies are subsequently quantified using Ig isotype-specific Sandwich and indirect ELISA, respectively. In order to correctly quantify the serum concentration of each Ig isotype, the samples need to be appropriately diluted to fit within the linear range of the standard curves. Using this protocol, we have consistently obtained reliable results with high specificity and sensitivity. When used in combination with other complementary methods such as flow cytometry, in vitro culture of splenic B cells and immunohistochemical staining (IHC), this protocol will allow researchers to gain a comprehensive understanding of antibody responses in a given experimental setting.

In this paper, we describe a protocol to characterize T-dependent and T-independent immunoglobulin (Ig) isotype responses in mice using ELISA. This method used alone or in combination with flow cytometry will allow researchers to identify differences in B cell-mediated Ig isotype responses in mice following T-dependent and T-independent antigen immunization.

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a ...

Imaging of In Situ Interferon Gamma Production in the Mouse Spleen following Listeria monocytogenes Infection

Cytokines are small proteins secreted by cells, mediating cell-cell communications that are crucial for effective immune responses. One characteristic of cytokines is their pleiotropism, as they are produced by and can affect a multitude of cell types. As such, it is important to understand not only which cells are producing cytokines, but also in which environment they do so, in order to define more specific therapeutics. Here, we describe a method to visualize cytokine production in situ following bacterial infection. This technique relies on imaging cytokine-producing cells in their native environment by confocal microscopy. To do so, tissue sections are stained for markers of multiple cell types together with a cytokine stain. Key to this method, cytokine secretion is blocked directly in vivo before harvesting the tissue of interest, allowing for detection of the cytokine that accumulated inside the producing cells. The advantages of this method are multiple. First, the microenvironment in which cytokines are produced is preserved, which could ultimately inform on the signals required for cytokine production and the cells affected by those cytokines. In addition, this method gives an indication of the location of the cytokine production in vivo, as it does not rely on artificial in vitro re-stimulation of the producing cells. However, it is not possible to simultaneously analyze cytokine downstream signaling in cells that receive the cytokine. Similarly, the cytokine signals observed correspond&#160;only to the time-window during which cytokine secretion was blocked. While we describe the visualization of the cytokine Interferon (IFN) gamma in the spleen following mouse infection by the intracellular bacteria Listeria monocytogenes, this method could potentially be adapted to the visualization of any cytokine in most organs.

Imaging of In Situ Interferon Gamma Production in the Mouse Spleen following Listeria monocytogenes Infection

Here, we describe a simple confocal imaging method to visualize the in situ localization of cells secreting the cytokine Interferon gamma in murine secondary lymphoid organs. This protocol can be extended for the visualization of other cytokines in diverse tissues.

Cytokines are small proteins secreted by cells, mediating cell-cell communications that are crucial for effective immune responses. One characteristic of ...

Evaluation of T Follicular Helper Cells and Germinal Center Response During Influenza A Virus Infection in Mice

T Follicular Helper (Tfh) cells are an independent CD4+&#160;T cell subset specialized in providing help for germinal center (GC) development and generation of high-affinity antibodies. In influenza virus infection, robust Tfh and GC B cell responses are induced to facilitate effective virus eradication, which confers a qualified mouse model for Tfh-associated study. In this paper, we described protocols in detection of basic Tfh-associated immune response during influenza virus infection in mice. These protocols include: intranasal inoculation of influenza virus; flow cytometry staining and analysis of polyclonal and antigen-specific Tfh cells, GC B cells and plasma cells; immunofluorescence detection of GCs; enzyme-linked immunosorbent assay (ELISA) of influenza virus-specific antibody in serum. These assays basically quantify the differentiation and function of Tfh cells in influenza virus infection, thus providing help for studies in elucidating differentiation mechanism and manipulation strategy.

This paper describes protocols of evaluating Tfh and GC B response in mouse model of influenza virus infection.

T Follicular Helper (Tfh) cells are an independent CD4+&#160;T cell subset specialized in providing help for germinal center (GC) development and ...

Retroviral Overexpression of CXCR4 on Murine B-1a Cells and Adoptive Transfer for Targeted B-1a Cell Migration to the Bone Marrow and IgM Production

As cell function is influenced by niche-specific factors in the cellular microenvironment, methods to dissect cell localization and migration can provide further insight on cell function. B-1a cells are a unique B cell subset in mice that produce protective natural IgM antibodies against oxidation-specific epitopes that arise during health and disease. B-1a cell IgM production differs depending on B-1a cell location, and therefore it becomes useful from a therapeutic standpoint to target B-1a localization to niches supportive of high antibody production. Here we describe a method to target B-1a cell migration to the bone marrow by retroviral-mediated overexpression of the C-X-C motif chemokine receptor 4 (CXCR4). Gene induction in primary murine B cells can be challenging and typically yields low transfection efficiencies of 10-20% depending on technique. Here we demonstrate that retroviral transduction of primary murine B-1a cells results in 30-40% transduction efficiency. This method utilizes adoptive cell transfer of transduced B-1a cells into B cell-deficient recipient mice so that donor B-1a cell migration and localization can be visualized. This protocol can be modified for other retroviral constructs and can be used in diverse functional assays post-adoptive transfer, including analysis of donor cell or host cell phenotype and function, or analysis of soluble factors secreted post B-1a cell transfer. The use of distinct donor and recipient mice differentiated by CD45.1 and CD45.2 allotype and the presence of a GFP reporter within the retroviral plasmid could also enable detection of donor cells in other, immune-sufficient mouse models containing endogenous B cell populations.

Here we describe a method for retroviral overexpression and adoptive transfer of murine B-1a cells to examine in vivo B-1a cell migration and localization. This protocol can be extended for diverse downstream functional assays including quantification of donor B-1a cell localization or analysis of donor cell-derived secreted factors post-adoptive transfer.

As cell function is influenced by niche-specific factors in the cellular microenvironment, methods to dissect cell localization and migration can provide ...

Isolating Malignant and Non-Malignant B Cells from lck:eGFP Zebrafish

Zebrafish (Danio rerio) are a powerful model to study lymphocyte development. Like mammals, D. rerio possess an adaptive immune system that includes B and T lymphocytes. Studies of zebrafish lymphopoiesis are difficult because antibodies recognizing D. rerio cell surface markers are generally not available, complicating isolation and characterization of different lymphocyte populations, including B-lineage cells. Transgenic lines with lineage-specific fluorophore expression are often used to circumvent this challenge. The transgenic lck:eGFP line has been used to study D. rerio T cell development, and has also been utilized to model T cell development and acute lymphoblastic leukemia (T-ALL). Although lck:eGFP fish have been widely used to analyze the T-lineage, they have not been used to study B cells. Recently, we discovered that many zebrafish B cells also express lck, albeit at lower levels. Consequently, lck:eGFP B cells likewise express low levels of GFP. Based on this finding, we developed a protocol to purify B-lineage cells from lck:eGFP zebrafish, which we report here. Our method describes how to utilize a fluorescent-activated cell sorter (FACS) to purify B cells from lck:eGFP fish or related lines, such as double-transgenic rag2:hMYC; lck:eGFP fish. In these lines, B cells, particularly immature B cells, express GFP at low but detectable levels, allowing them to be distinguished from T cells, which express GFP highly. B cells can be isolated from marrow, thymus, spleen, blood, or other tissues. This protocol provides a new method to purify D. rerio B cells, enabling studies focused on topics like B cell development and B lymphocyte malignancies.

Isolating Malignant and Non-Malignant B Cells from lck:eGFP Zebrafish

Transgenic lck:eGFP zebrafish express GFP highly in T lymphocytes, and have been used to study T cell development and acute lymphoblastic leukemia. This line can be used to study B cells, which express lck at lower levels. This protocol describes purification of malignant and non-malignant B cells from lck:eGFP zebrafish.

Zebrafish (Danio rerio) are a powerful model to study lymphocyte development. Like mammals, D. rerio possess an adaptive immune system that includes B and ...

Medicine

Isolation of Group 2 Innate Lymphoid Cells from Mouse Nasal Mucosa to Detect the Expression of CD226

With abundant research on group 2 innate lymphoid cells (ILC2s) published over the years, ILC2s are widely known to be implicated in regulating various pathological processes, including anti-helminth immunity, tissue repair, thermogenesis, and autoimmune diseases such as asthma and allergic rhinitis (AR). ILC2s permanently reside in peripheral tissues such as the skin, gut, lungs, and nasal cavity; however, there is limited information about their exact functions in nasal mucosal immunity. CD226 is an activating costimulatory molecule, mainly expressed on natural killer (NK) cells, T cells, and inflammatory monocytes. However, whether ILC2s express CD226 or play a role in the pathogenesis of ILC2s-related diseases remains unknown. Here, we established a method to isolate and identify ILC2s from the nasal mucosa and detected CD226 expression on ILC2s obtained from healthy and AR mice. Herein, we describe this protocol for the isolation and identification of ILC2s from mouse nasal mucosa, which will help explore the internal pathological mechanism of immunological disorders in nasal mucosal diseases.

Group 2 innate lymphoid cells (ILC2s), implicated in type 2 inflammation, mainly participate in response to helminth infection, allergic diseases, metabolic homeostasis, and tissue repair. In this study, a procedure to isolate ILC2s from murine nasal mucosa and detect the expression of CD226 is demonstrated.

With abundant research on group 2 innate lymphoid cells (ILC2s) published over the years, ILC2s are widely known to be implicated in regulating various ...

Analysis of Granulocyte-Macrophage Colony-Stimulating Factor-Producing T Helper Cells in a Mouse Model of Contact Hypersensitivity

Parallel to traditional Th1/Th2/Th17/Treg lineages, granulocyte-macrophage colony-stimulating factor-producing T helper (Th-GM) cells have been identified as a distinct subset of T helper cells (GM-CSF+ IFN-&#947;- IL-17A- IL-22- effector CD4+ T cells) in human and mice. Contact hypersensitivity (CHS) is considered an excellent animal model for allergic contact dermatitis (ACD) in human, manifesting an intact T cell-mediated immune response. To provide a standardized and comprehensive assay to analyze the Th-GM cell subset in the T cell-dependent immune response in vivo, a murine CHS model was induced by sensitization/challenge with a reactive, low-molecular-weight, organic hapten, 2,4-dinitrofluorobenzene (DNFB). The Th-GM subset in effector CD4+ T cells generated upon immunization with the hapten was analyzed by flow cytometry. We found that Th-GM was mainly expanded in lesions and draining lymph nodes in the DNFB-induced CHS mouse model. This method can be applied to further study the biology of Th-GM cells and pharmacological research of therapeutic strategies centered on GM-CSF in various conditions, such as ACD.

Here, we present a simple and standardized method of analyzing the granulocyte-macrophage colony-stimulating factor-producing T helper subset in vivo.

Parallel to traditional Th1/Th2/Th17/Treg lineages, granulocyte-macrophage colony-stimulating factor-producing T helper (Th-GM) cells have been identified ...

Detection of True IgE-expressing Mouse B Lineage Cells

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