This work was supported in part by the Lupus Research Institute, NCI grant CA138617, NIDDK grant DK090978, Charlotte Schmidlapp Award (to E.M.J.), and the Albert J. Ryan Fellowship (to J.K.). We are grateful for the support and instrumentation provided by the Research Flow Cytometry Core in the Division of Rheumatology at Cincinnati Children&#8217;s Hospital Medical Center, supported in part by NIH AR-47363, NIH DK78392 and NIH DK90971.

Animal work was performed under specific pathogen-free conditions in accordance with guidelines set by the Association for Assessment and Accreditation of Laboratory Animal Care International and our Institutional Animal Care and Use Committee (IACUC).
NOTE: Incorporate mice expressing a congenic marker such as CD45.1 on either donor or recipient animals if possible, because this allows for the monitoring of donor graft efficiency and specific expansion of the donor CD4 T cell population. If considering the use of otherwise genetically modified mice as donor or recipients, ensure the strain is properly backcrossed to the B6 background, or transferred cells may be rejected&#8212;this will be addressed in greater detail in the representative results and discussion sections.
NOTE: The following procedures detail volumes for harvesting 4 donor bm12 mice, which should yield enough cells to inject 12-16 mice. Six to twelve week-old bm12 mice yield roughly 100-140 million lymphocytes with approximately 25% CD4 T cells. If starting with different numbers of mice, or different mouse strains, scale volumes up or down accordingly. C57BL/6 mice generally give similar yields with closer to only 20% CD4 T cells.
NOTE: Perform all steps at RT and use RT media to avoid heat and cold shock, which can impair long-term lymphocyte viability. Perform tissue-harvesting and tissue-processing steps in a tissue culture hood using aseptic technique. All media in this protocol is IMDM with 10% heat-inactivated FBS, unless indicated otherwise, and will be simply referred to as &#8220;complete media.&#8221;
1. Novel Method for Genotyping bm12 Mice
NOTE: A brief restriction digest-based protocol for genotyping is provided here, as a simple and inexpensive alternative to sequencing, which is currently the only published genotyping method for these mice.
<ol>
	<li>Isolate genomic DNA from mouse tails. Please refer to a previous JoVE article for detailed protocols on mouse tail clipping and the generation of cDNA from tail digests13.</li>
	<li>Perform a reverse-transcriptase polymerized chain reaction (PCR)14 to amplify a common 474 bp DNA fragment from MHC-II I-Ab and I-Abm12 using primers and thermocycling conditions listed in Table 1.</li>
	<li>Perform restriction digest on ~7 &#181;l of the PCR product using the enzyme PsuI, or one of its isoschizomers (BstX2I, BstYI, MflI, or XhoII), which cuts wild type I-Ab, but not I-Abm12 mutant. Follow digest protocol provided by the manufacturer, which will specify a mixture of water, buffer and enzyme, and an incubation of 5 min. to several hours at 37 &#176;C15.</li>
	<li>Load digest product and run on 1% polyacrylamide gel with ethidium bromide14 for 30-45 min. at 150V&#8212;though optimal voltage and time settings will vary depending on the PCR gel apparatus used. Visualize DNA bands with a UV illuminator. Representative results are shown in Figure 1.</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/53319/53319fig1.jpg" /> 
Figure 1. Representative bm12 genotyping results. 
To identify mice homozygous for I-Abm12/bm12, tail DNA was screened for bm12 by PCR/restriction digest genotyping (Step 1). Homozygous wild type (I-Ab/b) DNA yields two bands at 227 and 247 bp (visualized as one thick band at ~250 bp); homozygous bm12 (I-Abm12/bm12) DNA yields one band at 474 bp; and heterozygous (I-Abm12/b) DNA yields two bands at ~250 and 474 bp. <a href="https://www.jove.com/files/ftp_upload/53319/53319fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. Harvesting Donor Cells
<ol>
	<li>Sacrifice donor mice using Institutional Animal Care and Use Committee (IACUC)-approved primary and secondary euthanasia methods. For example, euthanize mice by asphyxiation with CO2 followed by cervical dislocation.</li>
	<li>Using aseptic technique, harvest spleens and lymph nodes (superficial cervical, mandibular, brachial, axillary, mesenteric, and inguinal) into 15 ml tubes containing 10 ml complete media as in11-13. 
	NOTE: Refer to previous reports for detailed lymph node dissection protocols16&#8211;18. This model is also successful using only mouse splenocytes for transfer, but the addition of lymph nodes significantly reduces the required number of donor mice.</li>
	<li>Generate a single-cell suspension by mashing lymphoid tissues collected in step 2.2 through 70 &#181;m cell strainers using the plunger from a syringe. For better yields, do not overload strainers&#8212;use ~6 strainers for every 4 mice processed, and rinse strainers frequently while processing. Combine tissues from 4 mice into two 50 ml conical tubes, then centrifuge cells for 5 min at 400 x g and decant supernatant.</li>
	<li>Resuspend cells from both conical tubes in 50 ml complete media and transfer to new conical tube. Keep this tube at RT. 
	NOTE: Fat adheres to the sides of the tube, and if the tube is not replaced, final cellular yields can suffer.</li>
</ol>
3. Donor Cell Counting
NOTE: To preserve the highest viability, red blood cell (RBC) lysis of the entire sample is not recommended.
<ol>
	<li>Mix single cell suspension from step 2.4 well, remove 1 ml and transfer it to a separate 50 ml conical. Set aside remaining, untouched cells (49 ml) at RT while counting.</li>
	<li>Add 3 ml of an ammonium chloride-based red blood cell lysis buffer to the 1 ml donor cell sample. Mix gently for 1 min. by rocking, then fill to 50 ml with complete media, and centrifuge for 5 min. at 400 x g and decant supernatant.</li>
	<li>Resuspend RBC-lysed cells in 10 ml complete media and count (e.g., with trypan blue using a hemocytometer). Multiply result by 49&#8212;this is the total number of cells remaining in the non-lysed sample that was set aside. Discard RBC-lysed cells.</li>
</ol>
4. Donor Cell Labeling and/or CD4 T Cell Purification
<ol>
	<li>If desired, purify CD4 T cells at this stage, though this is not necessary. Additionally, if desired, label cells with CFSE, as in19, or other cell tracking dyes, an example of which is shown in Figure 4 of the representative results section. 
	NOTE: For CD4 T cell purification, negative magnetic selection is recommended, as it can achieve high purity and leaves cells untouched with high viability as described in20. It is important that endotoxin-free buffers are used; therefore, instead of BSA, make separation buffer with 2% FBS and the recommended concentration of EDTA.</li>
</ol>
5. Injection of Donor bm12 Cells
<ol>
	<li>After counting donor lymphocytes in step 3 (and purified or labeled as in step 4, as desired), centrifuge cells for 5 min&#160;at 400 x g. Decant supernatant and resuspend cells in PBS at 120 million lymphocytes per ml (or 30 million purified CD4 T cells per ml). Transfer cells to a sterile 5 ml round-bottom tube, or other sterile tube that easily accommodates a 1 ml syringe fitted with a 27.5 G x 13 mm needle.</li>
	<li>Prior to injection, set aside a small sample of donor cells from step 5.1 at 4 &#176;C for flow staining to determine the percentage of CD4 T cells within donor samples. Stain these samples as described in Step 8 using the minimal antibody panel (Table 2). 
	NOTE: If cells from different mouse strains are used as separate donors, this is an important consideration, and if CD4 T cell percentages vary substantially, purification may be required.</li>
	<li>Mix cells gently, but thoroughly. This can be done by pipetting cells up and down using a 1 ml syringe without an attached needle. After mixing, draw cells into the 1 ml syringe. Attach needle after removing any air bubbles. Keeping the needle off while priming the syringe helps maintain cell viability.</li>
	<li>Inject 250 &#181;l per mouse (which is equal to 30 million lymphocytes, or 7.5 million purified CD4 T cells per mouse) intraperitoneally, as described in21. 
	NOTE: In experiments shown here and in our prior work11, each mouse is injected with 30 million total lymphocytes from bm12 donors, rather than the 100 million total splenocytes traditionally used. In unpublished data from our lab, no difference was observed in serum anti-dsDNA at day 14 following injections of 30 or 100 million lymphocytes per mouse. While this significantly reduces the number of mice needed for experiments, the development of nephritis with this number of cells has not been assessed.</li>
</ol>
6. Determine Grafting Efficiency
NOTE 1: This section will describe how to determine the degree of donor cell grafting in the recipient at day 3 in order to identify any mice which may have received suboptimal injections (e.g.,&#160;the graft in one mouse is &#60;10% of that seen in all other mice from the same group). These data can also help determine whether cells from a genetically modified mouse strain are rejected at later time points (for details, see representative results section, Figure 6).
NOTE 2: This section is only possible if donors and recipients are from mice on different congenic backgrounds, e.g., when using CD45.1 bm12 donors and CD45.2 C57BL/6 recipients, or when donor cells are labeled with a cell tracking dye. Importantly, at 3 days post-injection, CD4 T cells have undergone minimal expansion (see representative results section, Figure 4), so differences observed in the degree of grafting are due to variability in injections, not expansion.
<ol>
	<li>Anesthetize mice with 4% isoflurane or other IACUC-approved method. Test rear foot reflexes to ensure mouse is properly anesthetized before proceeding to the blood draw.</li>
	<li>Harvest 100-200 &#181;l blood using an IACUC-approved method, such as retro-orbital puncture as in22. Collect blood into individual 0.5 ml microcentrifuge tubes containing an anti-coagulant, such as the plasma collection tubes referenced in the materials table, which contain lyophilized dipotassium EDTA. After blood collection, apply gentle pressure to the mouse eye using a sterile towel or gauze to ensure the bleeding stops, then place the mouse back in its home cage where it will remain until the final tissue harvest (Step 7). 
	NOTE: Do not leave mice unattended until mice have regained sufficient consciousness to maintain ventral recumbency, and do not return mice to the company of others until fully recovered.</li>
	<li>Bring blood volume to 500 &#181;l with 21 &#176;C PBS and transfer to conical-bottom microcentrifuge tube, then slowly underlay 200 &#181;l of 21 &#176;C high density cell separation solution with a 200 &#181;l pipet, being careful to minimize mixing between the two phases (see Materials Table for recommended solutions). Centrifuge cells at 700 x g for 20 min at 20-25 &#176;C with centrifuge brake set to low.</li>
	<li>Remove top layer containing lymphocytes with a 1 ml pipet and transfer to a new microcentrifuge tube containing 800 &#181;l cold complete media. Gently vortex to mix cells. Centrifuge cells at 700 x g for 5 min at 4 &#176;C and decant supernatant.</li>
	<li>Resuspend cells in 200 &#181;l complete media, transfer to a 96-well U-bottom plate, and stain for flow cytometry as described in Step 8 using a minimal antibody panel (Table 2) to determine the relative abundance of the CD4 T cell graft as a percentage of PBMCs.</li>
</ol>
7. Final Tissue Harvest
NOTE: The experiments described in the results section were harvested 14 days after injection of donor cells (or in some cases less time), as they focus on the initial development of Tfh cells and plasma cells; however, since this model is a chronic GVHD model of SLE, disease can be monitored at much later time points. The optimal timeframe will depend upon the research question posed in each individual experiment.
<ol>
	<li>At a predetermined time point after injection of bm12 donor cells (step 5.4), sacrifice mice using an IACUC-approved method of euthanasia. Asphyxiation with CO2 followed by exsanguination is recommended. Cervical dislocation can function as a secondary method of euthanasia, but this may reduce the blood draw yield.</li>
	<li>Wet the abdomen of the mouse lightly with a spray bottle containing 70% ethanol. Make a small, superficial incision with surgical scissors approximately 1 cm above the genitalia. Draw back the skin of the abdomen toward the sternum, being careful to keep the peritoneal fascia intact. 
	NOTE: Diseased mice usually present with 0.5-3 ml ascites at day 14, which, although it has not yet been well characterized, can be measured and analyzed as an additional disease parameter.</li>
	<li>Fit a 5 ml syringe with an 18 G&#160;needle. Insert the needle into the lower right quadrant of the abdomen with the needle directed up toward the animal&#8217;s head and at 15&#176; angle to the plane of the fascia. Position the needle tip near the cecum to help prevent the needle from getting clogged with intestine while aspirating ascites.</li>
	<li>Carefully rotate the mouse on its side, then slowly draw ascites into the syringe. Once ascites has been recovered, remove the syringe and record the aspirate volume, based upon the volumetric markings on the side of the syringe.</li>
	<li>Discharge ascites into a 5 ml round-bottom tube and store on ice for later processing.</li>
	<li>Harvest blood via draw from the inferior vena cava (IVC) essentially as in22. Using dull forceps, gently move intestines to the left side of the mouse, uncovering the IVC. Insert a 27.5 G&#160;needle fitted to a 1 ml syringe into the IVC and slowly draw 400-500 &#181;l of blood.</li>
	<li>To minimize hemolysis, slowly inject blood into a 0.5 ml microcentrifuge serum or plasma collection tube. For later serum analysis of ANAs by ELISA, keep blood on ice.</li>
	<li>Dissect and obtain additional relevant tissues (spleen and, if desired, lymph nodes and kidneys, particularly if collecting at later time points and glomerulonephritis will be scored). Remove spleen by gently placing the intestines back toward the right side of the animal, and pulling on the pancreas, which is the spleen&#8217;s primary connective tissue.</li>
	<li>Remove any pancreas remaining, then weigh spleens on a high precision balance immediately after dissection, as gross splenomegaly is a commonly reported parameter in mouse models of SLE. Place lymphoid tissues into individual 1.5 ml tubes filled with 1ml complete media on ice. Fix kidneys in 10% neutral buffered formalin or snap freeze kidneys for later histology as in23.</li>
	<li>Centrifuge blood within 2 hr of collection for 3 min. at 10,000 x g (4 &#176;C). Remove serum and store at -80 &#176;C for later analysis of ANA by ELISA. Refer to previous reports for detailed ANA ELISA protocols24,25. Store serum in multiple 10-20 &#181;l aliquots to minimize freeze/thaw cycles, and allow a greater number of future assays.</li>
	<li>Centrifuge ascites 400 x g for 5 min. Remove the supernatant with a 1 ml pipet and aliquot into several 0.5 ml tubes. Freeze supernatant at -80 &#176;C for later analyses of anti-nuclear antibodies (ANAs) or other soluble inflammatory mediators.</li>
	<li>Resuspend cellular fraction in approximately 1 ml complete media and transfer 200 &#181;l to a 96-well U-bottom plate for flow staining (as in Step 8).</li>
	<li>Mash each spleen through 70 &#181;m cell strainers into separate tubes and rinse with complete media. Resuspend splenocytes with 1 ml cold RBC lysis buffer for 1 min. and mix gently by rocking. Bring volume to 10 ml with cold complete media and centrifuge for 5 min at 400 x g and decant supernatant.</li>
	<li>Resuspend cell pellet in 5 ml complete media and count using a hemocytometer. Adjust volume such that 200 &#181;l of complete media contains 1-3 million cells. Begin staining for flow cytometry (Step 8) using an extended panel (Table 2) to quantify donor CD4 T cell and recipient B cell differentiation and expansion. 
	NOTE: Depending upon what laser, photomultiplier tube (PMT), and filter set combinations are available, separating the T and B cell analysis panel into multiple panels may be necessary.</li>
</ol>
8. Flow Staining
<ol>
	<li>Transfer 1-3 million splenocytes in 200 &#181;l complete media into individual 5 ml round-bottom tubes, or into separate wells of a 96-well U-bottom plate. 
	NOTE: Using a 96-well plate is an efficient way to stain multiple samples, but take care to plate samples in every other well in order to prevent cross-contamination. One 96-well plate can accordingly hold 24 samples.</li>
	<li>Centrifuge plate at 500 x g for 3 min., then flick supernatant from plate into appropriate (biohazard) container.</li>
	<li>Resuspend the cell pellets with 100 &#181;l of flow buffer (1% FBS in PBS) containing a fixable viability dye at the manufacturer&#8217;s recommended concentration and purified anti-CD16/anti-CD32 antibody cocktail at 1 &#181;g/ml. Incubate for 10 min at 20-25 &#176;C, then add 100 &#181;l cold complete media to quench viability dye. 
	NOTE: Splenocytes from diseased mice usually contain relatively high numbers of dead or dying cells; therefore, the inclusion of a viability dye is recommended to avoid non-specific antibody labeling of dying cells, resulting in cleaner, more reliable data. Similarly, the anti-CD16/anti-CD32 cocktail is included to block non-specific fluorescently-labeled antibody binding by Fc receptors.</li>
	<li>Centrifuge plate at 500 x g for 3 min., then flick supernatant from plate into biohazard container. Resuspend cells in 200 &#181;l flow buffer. Centrifuge plate at 500 x g for 3 min., then flick supernatant from plate into biohazard container.</li>
	<li>Resuspend cells in 50 &#181;l flow buffer containing antibody cocktail (Table 2). Incubate for 20 min at 4 &#176;C, then add 150 &#181;l flow buffer. Repeat wash step 8.4.</li>
	<li>Resuspend cells with 100 &#181;l 2% paraformaldehyde in PBS. Incubate for 30 min at 4 &#176;C, then add 100 &#181;l flow buffer. Centrifuge plate at 500 x g for 3 min., then flick supernatant from plate into biohazard container.</li>
	<li>Resuspend in 200 &#181;l flow buffer, transfer to flow tube inserts or standard flow tubes, add an additional 100-200 &#181;l flow buffer, and store at 4 &#176;C in the dark until acquiring on flow cytometer.</li>
	<li>Acquire on a flow cytometer equipped with the appropriate lasers and PMTs for the chosen antibody panels within several days of staining. Record forward scatter width and/or height in addition to forward scatter area, side scatter area, and area of the fluorescent parameters utilized. For reliable results, acquire &#8805;1,000 donor cells in each WT sample, or an equivalent number of total lymphocytes in genetically modified or otherwise manipulated mice that show minimal expansion of donor cells, where collection of so many events may not be feasible. 
	NOTE: Flow cytometry analyses are described in detail in the representative results section (Figures 3-6).</li>
</ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+caveat+for+T+cell+transfer+studies:+generation+of+cytotoxic+anti-Thy1.2+antibodies+in+Thy1.1+congenic+mice+given+Thy1.2++tumors+or+T+cells.">A caveat for T cell transfer studies: generation of cytotoxic anti-Thy1.2 antibodies in Thy1.1 congenic mice given Thy1.2+ tumors or T cells.</a> J Leukoc Biol. 89 (2), 291-300 (2011).</li><li>Scott, D. M., Ehrmann, I. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+mouse+male-specific+transplantation+antigen+H-Y..">Identification of a mouse male-specific transplantation antigen H-Y..</a> Nature. 376, 695-698 (1995).</li><li>Joly, E., Hudrisier, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+is+trogocytosis+and+what+is+its+purpose.">What is trogocytosis and what is its purpose.</a> Nat Immunol. 4 (9), 815 (2003).</li><li>Brown, D. R., Calpe, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutting+edge:+an+NK+cell-independent+role+for+Slamf4+in+controlling+humoral+autoimmunity.">Cutting edge: an NK cell-independent role for Slamf4 in controlling humoral autoimmunity.</a> J Immunol. 187 (1), 21-25 (2011).</li><li>Morris, S. C., Cheek, R. L., Cohen, P. L., Eisenberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allotype-specific+immunoregulation+of+autoantibody+production+by+host+B+cells+in+chronic+graft-versus+host+disease.">Allotype-specific immunoregulation of autoantibody production by host B cells in chronic graft-versus host disease.</a> J Immunol. 144 (3), 916-922 (1990).</li><li>Choudhury, A., Cohen, P. L., Eisenberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B+cells+require+&#8220;nurturing&#8221;+by+CD4+T+cells+during+development+in+order+to+respond+in+chronic+graft-versus-host+model+of+systemic+lupus+erythematosus.">B cells require &#8220;nurturing&#8221; by CD4 T cells during development in order to respond in chronic graft-versus-host model of systemic lupus erythematosus.</a> Clin Immunol. 136 (1), 105-115 (2010).</li><li>Slifka, M. K., Antia, R., Whitmire, J. K., Ahmed, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Humoral+immunity+due+to+long-lived+plasma+cells.">Humoral immunity due to long-lived plasma cells.</a> Immunity. 8 (3), 363-372 (1998).</li></ol>

The authors have nothing to disclose.

The bm12 inducible model is a relatively easy and efficient way to study the cellular and molecular processes of SLE. Chronic activation of adoptively transferred CD4 T cells directed against self antigens leads to the accumulation of Tfh, GC B cells, and plasma cells which can be measured by flow cytometry, as described here. Future studies using this model can quickly and easily interrogate the role of candidate genes and novel therapies in the autoimmune germinal center processes which resemble those occurring in pati...

Systemic lupus erythematosus (SLE) is a complex autoimmune disease characterized prototypically by anti-nuclear antibody (ANA) production and glomerulonephritis. Numerous other sequelae, including dermal, cardio-pulmonary, and hepatic lesions are associated with disease in some individuals. Prevalence estimates in the US vary widely, from 150,000-1,500,0001,2, with particularly high incidence in women and minorities3. Although the etiology of SLE has been difficult to discern, it is thought to arise from the interplay of various genetic and environmental factors, which culminate in systemic autoimmunity.
Numerous animal models have been employed to study factors leading to disease onset and progression. Classic mouse models of SLE include genetically predisposed mouse strains including the NZB x NZW F1 model and its NZM derivatives, the MLR/lpr strain, and the BXSB/Yaa strain, and inducible systems, such as the pristane and chronic graft-versus-host disease (cGVHD) models4. Early reports of autoantibody production in GVHD models used various mouse strains or hamster strains for parent into F1 transfers5&#8211;8; more common methods used to study lupus-like disease currently include the DBA/2 parent&#8594;(C57BL/6 x DBA/2) F1, and the bm12 transfer model described here. Each model has its own caveats, but they generally share a common set of features that correlate with clinical features of human disease. The most often reported parameters in mouse models include splenomegaly, lymphadenopathy, nephritis, ANA production, and at the cellular level, the expansion of T follicular helper cells (Tfh), germinal center (GC) B cells, and plasma cells.
The inducible bm12 model is achieved by the adoptive transfer of lymphocytes from I-Abm12B6(C)-H2-Ab1bm12/KhEgJ (bm12) mice, a strain identical to C57BL/6 except for 3 amino acid substitutions on MHC class II, into I-Ab C57BL/6 (B6) mice. Alloactivation of donor CD4 T cells by recipient APCs leads to cGVHD with symptoms closely resembling SLE. Specifically, these include expansion of donor-derived Tfh, expansion of recipient-derived GC B cells and plasma cells, and production of ANAs including anti-dsDNA, anti-ssDNA, anti-chromatin, and anti-RBC antibodies9. Over time, recipient mice develop glomerulonephritis associated with IgG deposits in the glomerular, interstitial, and vascular regions of the kidneys10. We have recently shown that, similar to human disease, there is also a critical role for type I IFN in this model11. Notably, the defining criteria for human SLE include the development of nephritis compatible with SLE in the presence of anti-dsDNA antibodies12, both of which are prominent features of this mouse model.
There are several advantages of the bm12 model over the spontaneous models. Classic models that develop SLE-like signs spontaneously rely upon either hybrid mouse strains, inbred mouse strains not on the B6 background, or large genetic loci on the B6 background, which make crossing to knockout or otherwise genetically modified mice difficult and time consuming. With the bm12 inducible model, genetically modified mice can serve as either the donor or recipient, allowing more rapid identification of the cellular compartment in which particular genes may be important for disease. Furthermore, disease development in the bm12 model is much faster, requiring only 2 weeks until the appearance of ANAs, compared to several months for most spontaneous models. Moreover, in contrast to the spontaneous models that develop disease at different time points, the disease onset and progression in the bm12&#8594;B6 model is highly synchronized. This allows for the generation of appropriately sized cohorts that can be used for interventional or therapeutic strategies at any stage of disease development.
What follows is a detailed protocol for initiating SLE-like autoimmunity by the adoptive transfer of bm12 lymphocytes into C57BL/6 mice, or genetic variants on the B6 background. Additionally, we describe a flow cytometric staining protocol for enumerating Tfh, GC B cells, and plasma cells&#8212;cell types associated with human disease. Importantly, these protocols can also be used to characterize disease in most mouse models of SLE and identify Tfh, GC B cells and plasma cells in other disease models.

<table border="0">
	<tbody>
		<tr>
			<td>B6.SJL-Ptprca Pepcb/BoyJ</td>
			<td>The Jackson Laboratory</td>
			<td>001162</td>
			<td>CD45.1+ BoyJ mouse strain</td>
		</tr>
		<tr>
			<td>B6(C)-H2-Ab1bm12/KhEgJ</td>
			<td>The Jackson Laboratory</td>
			<td>001162</td>
			<td>Bm12 mouse strain</td>
		</tr>
		<tr>
			<td>FastDigest PsuI</td>
			<td>Life Technologies</td>
			<td>FD1554</td>
			<td>Restriction digest enzyme for genotyping</td>
		</tr>
		<tr>
			<td>1X RBC Lysis Buffer</td>
			<td>eBioscience</td>
			<td>00-4333-57</td>
			<td></td>
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		<tr>
			<td>IMDM</td>
			<td>GE Healthcare</td>
			<td>SH30228.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasma Separation Tube (PST)</td>
			<td>BD</td>
			<td>365974</td>
			<td>Blood collection tube with Dipotassium EDTA</td>
		</tr>
		<tr>
			<td>Serum Separation Tube (SST)</td>
			<td>BD</td>
			<td>365967</td>
			<td>Blood collection tube with Clot activator / SST Gel</td>
		</tr>
		<tr>
			<td>Ficoll</td>
			<td>GE Healthcare</td>
			<td>17-1440-02&#160;</td>
			<td>High density cell separation solution</td>
		</tr>
		<tr>
			<td>Lympholyte-M</td>
			<td>Cedarlane</td>
			<td>CL5030</td>
			<td>High density cell separation solution</td>
		</tr>
		<tr>
			<td>GL-7-biotin</td>
			<td>eBioscience</td>
			<td>13-5902-82&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin-BUV395</td>
			<td>BD</td>
			<td>564176</td>
			<td></td>
		</tr>
		<tr>
			<td>CD138-BV421</td>
			<td>BioLegend</td>
			<td>142508</td>
			<td></td>
		</tr>
		<tr>
			<td>CD4-BV510</td>
			<td>BioLegend</td>
			<td>100559</td>
			<td></td>
		</tr>
		<tr>
			<td>TCR&#946;-BV605</td>
			<td>BD</td>
			<td>562840</td>
			<td></td>
		</tr>
		<tr>
			<td>CD45.1-BV711</td>
			<td>BioLegend</td>
			<td>110739</td>
			<td></td>
		</tr>
		<tr>
			<td>CD45.2-FITC</td>
			<td>BioLegend</td>
			<td>109806</td>
			<td></td>
		</tr>
		<tr>
			<td>PD-1-PE</td>
			<td>BioLegend</td>
			<td>135206</td>
			<td></td>
		</tr>
		<tr>
			<td>CD19-PerCP</td>
			<td>BioLegend</td>
			<td>115532</td>
			<td></td>
		</tr>
		<tr>
			<td>Fas-PE-Cy7</td>
			<td>BD</td>
			<td>557653</td>
			<td></td>
		</tr>
		<tr>
			<td>CXCR5-APC</td>
			<td>BioLegend</td>
			<td>145506</td>
			<td></td>
		</tr>
		<tr>
			<td>Fixable Viability Dye ef780</td>
			<td>eBioscience</td>
			<td>65-0865-18</td>
			<td></td>
		</tr>
		<tr>
			<td>CD4-BV421</td>
			<td>BioLegend</td>
			<td>100443</td>
			<td></td>
		</tr>
		<tr>
			<td>1.2 ml FACS tube inserts, racked</td>
			<td>USA Scientific</td>
			<td>1412-1400</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Falcon&#8482; Round-Bottom Tubes</td>
			<td>BD</td>
			<td>352017</td>
			<td></td>
		</tr>
	</tbody>
</table>

the bm12 inducible model of systemic lupus erythematosus (sle) in c57bl/6 mice

Systemic lupus erythematosus (SLE) is an autoimmune disease with diverse clinical and immunological manifestations. Several spontaneous and inducible animal models mirror common components of human disease, including the bm12 transfer model. Upon transfer of bm12 splenocytes or purified CD4 T cells, C57BL/6 mice rapidly develop large frequencies of T follicular helper cells (Tfh), germinal center (GC) B cells, and plasma cells followed by high levels of circulating anti-nuclear antibodies. Since this model utilizes mice on a pure C57BL/6 background, researchers can quickly and easily study disease progression in transgenic or knockout mouse strains in a relatively short period of time. Here we describe protocols for the induction of the model and the quantitation Tfh, GC B cells, and plasma cells by multi-color flow cytometry. Importantly, these protocols can also be used to characterize disease in most mouse models of SLE and identify Tfh, GC B cells, and plasma cells in other disease models.

Diseased mice develop splenomegaly in as little as 14 days, exhibiting spleens 2-3 times the size of healthy mice in terms of mass and cellularity (Figure 2).
Splenocytes are sequentially gated on light scatter (FSC-A by SSC-A), elimination of doublets (FSC-W or -H by FSC-A), viable cells (low staining of viability dye), and CD4+TCR&#946;+ (Figure 3A). Donor cells are distinguished from recipient cells based on CD45.1 and CD45.2 (...

Watch this Scientific Journal Video about The bm12 Inducible Model of Systemic Lupus Erythematosus SLE in C57BL/6 Mice at JoVE.com

The bm12 Inducible Model of Systemic Lupus Erythematosus SLE in C57BL/6 Mice

The transfer of bm12 lymphocytes into a C57BL/6 recipient is an established model of systemic lupus erythematosus. Here we describe how to initiate disease using this model and how to characterize T follicular helper cells, germinal center B cells and plasma cells by flow cytometry.

The bm12 Inducible Model of Systemic Lupus Erythematosus (SLE) in C57BL/6 Mice

Systemic lupus erythematosus (SLE) is an autoimmune disease with diverse clinical and immunological manifestations. Several spontaneous and inducible ...

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17.3K Views.  Cincinnati Children's Hospital Medical Center and University of Cincinnati College of Medicine. The transfer of bm12 lymphocytes into a C57BL/6 recipient is an established model of systemic lupus erythematosus. Here we describe how to initiate disease using this model and how to characterize T follicular helper cells, germinal center B cells and plasma cells by flow cytometry.

Video: The bm12 Inducible Model of Systemic Lupus Erythematosus SLE in C57BL/6 Mice

Transthoracic Echocardiography in Mice

In recent years, murine models have become the primary avenue for studying the molecular mechanisms of cardiac dysfunction resulting from changes in gene expression. Transgenic and gene targeting methods can be used to generate mice with altered cardiac size and function,1-3 and as a result, in vivo techniques are needed to evaluate their cardiac phenotype. Transthoracic echocardiography, pulse wave Doppler (PWD), and tissue Doppler imaging (TDI) can be used to provide dimensional measurements of the mouse heart and to quantify the degree of cardiac systolic and diastolic performance. Two-dimensional imaging is used to detect abnormal anatomy or movements of the left ventricle, whereas M-mode echo is used for quantification of cardiac dimensions and contractility.4,5 In addition, PWD is used to quantify localized velocity of turbulent flow,6 whereas TDI is used to measure the velocity of myocardial motion.7 Thus, transthoracic echocardiography offers a comprehensive method for the noninvasive evaluation of cardiac function in mice.

Transthoracic echocardiography offers a noninvasive method for the evaluation of cardiac function in mice. A combination of ultrasound and Doppler imaging modalities can be used to obtain dimensional measurements of the heart and intracardiac blood flow, which together provide an assessment of cardiac systolic and diastolic performance.

In recent years, murine models have become the primary avenue for studying the molecular mechanisms of cardiac dysfunction resulting from changes in gene ...

The Spared Nerve Injury (SNI) Model of Induced Mechanical Allodynia in Mice

Peripheral neuropathic pain is a severe chronic pain condition which may result from trauma to sensory nerves in the peripheral nervous system. The spared nerve injury (SNI) model induces symptoms of neuropathic pain such as mechanical allodynia i.e. pain due to tactile stimuli that do not normally provoke a painful response [1]. 
The SNI mouse model involves ligation of two of the three branches of the sciatic nerve (the tibial nerve and the common peroneal nerve), while the sural nerve is left intact [2]. The lesion results in marked hypersensitivity in the lateral area of the paw, which is innervated by the spared sural nerve. The non-operated side of the mouse can be used as a control. The advantages of the SNI model are the robustness of the response and that it doesn&#x2019;t require expert microsurgical skills.
The threshold for mechanical pain response is determined by testing with von Frey filaments of increasing bending force, which are repetitively pressed against the lateral area of the paw [3], [4]. A positive pain reaction is defined as sudden paw withdrawal, flinching and/or paw licking induced by the filament. A positive response in three out of five repetitive stimuli is defined as the pain threshold. 
As demonstrated in the video protocol, C57BL/6 mice experience profound allodynia as early as the day following surgery and maintain this for several weeks.

The Spared Nerve Injury SNI Model of Induced Mechanical Allodynia in Mice

The Spared Nerve Injury animal model is described here as a mouse model of peripheral neuropathic pain following partial denervation of the sciatic nerve by lesioning the tibial and common peroneal nerve branches, leaving the remaining sural nerve intact. Behavioral modification resulting from mechanical allodynia is quantified by von Frey filaments.

Peripheral neuropathic pain is a severe chronic pain condition which may result from trauma to sensory nerves in the peripheral nervous system. The spared ...

Tibial Nerve Transection - A Standardized Model for Denervation-induced Skeletal Muscle Atrophy in Mice

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of denervation-induced skeletal muscle atrophy in rodents. Although originally developed and used extensively in the rat due to its larger size, the tibial nerve in mice is big enough that it can be easily manipulated with either crush or transection, leaving the peroneal and sural nerve branches of the sciatic nerve intact and thereby preserving their target muscles. Thus, this model offers the advantages of inducing less morbidity and impediment of ambulation than the sciatic nerve transection model and also allows investigators to study the physiologic, cellular and molecular biologic mechanisms regulating the process of muscle atrophy in genetically engineered mice. The tibial nerve supplies the gastrocnemius, soleus and plantaris muscles, so its transection permits the study of denervated skeletal muscle composed of fast twitch type II fibers and/or slow twitch type I fibers. Here we demonstrate the tibial nerve transection model in the C57Black6 mouse. We assess the atrophy of the gastrocnemius muscle, as a representative muscle, at 1, 2, and 4 weeks post-denervation by measuring muscle weights and fiber type specific cross-sectional area on paraffin-embedded histologic sections immunostained for fast twitch myosin.

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of skeletal muscle atrophy. The model surgical protocol is described and demonstrated in C57Black6 mice. &#160;

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of denervation-induced skeletal muscle atrophy in rodents. ...

Assessing Changes in Volatile General Anesthetic Sensitivity of Mice after Local or Systemic Pharmacological Intervention

One desirable endpoint of general anesthesia is the state of unconsciousness, also known as hypnosis. Defining the hypnotic state in animals is less straightforward than it is in human patients. A widely used behavioral surrogate for hypnosis in rodents is the loss of righting reflex (LORR), or the point at which the animal no longer responds to their innate instinct to avoid the vulnerability of dorsal recumbency. We have developed a system to assess LORR in 24 mice simultaneously while carefully controlling for potential confounds, including temperature fluctuations and varying gas flows. These chambers permit reliable assessment of anesthetic sensitivity as measured by latency to return of the righting reflex (RORR) following a fixed anesthetic exposure. Alternatively, using stepwise increases (or decreases) in anesthetic concentration, the chambers also enable determination of a population's sensitivity to induction (or emergence) as measured by EC50 and Hill slope. Finally, the controlled environmental chambers described here can be adapted for a variety of alternative uses, including inhaled delivery of other drugs, toxicology studies, and simultaneous real-time monitoring of vital signs.

Loss of the righting reflex has long served as a standard behavioral surrogate for unconsciousness, also called hypnosis, in laboratory animals. Alterations in volatile anesthetic sensitivity caused by pharmacological interventions can be detected with a carefully controlled high-throughput assessment system, which may be adapted for delivery of any inhaled therapeutic.

One desirable endpoint of general anesthesia is  the state of unconsciousness, also known as hypnosis. Defining the hypnotic state in animals is  less ...

The Sciatic Nerve Cuffing Model of Neuropathic Pain in Mice

Neuropathic pain arises as a consequence of a lesion or a disease affecting the somatosensory system. This syndrome results from maladaptive changes in injured sensory neurons and along the entire nociceptive pathway within the central nervous system. It is usually chronic and challenging to treat. In order to study neuropathic pain and its treatments, different models have been developed in rodents. These models derive from known etiologies, thus reproducing peripheral nerve injuries, central injuries, and metabolic-, infectious- or chemotherapy-related neuropathies. Murine models of peripheral nerve injury often target the sciatic nerve which is easy to access and allows nociceptive tests on the hind paw. These models rely on a compression and/or a section. Here, the detailed surgery procedure for the &#34;cuff model&#34; of neuropathic pain in mice is described. In this model, a cuff of PE-20 polyethylene tubing of standardized length (2 mm) is unilaterally implanted around the main branch of the sciatic nerve. It induces a long-lasting mechanical allodynia, i.e., a nociceptive response to a normally non-nociceptive stimulus that can be evaluated by using von Frey filaments. Besides the detailed surgery and testing procedures, the interest of this model for the study of neuropathic pain mechanism, for the study of neuropathic pain sensory and anxiodepressive aspects, and for the study of neuropathic pain treatments are also discussed.

Neuropathic pain is a consequence of a lesion or disease affecting the somatosensory system. The &#8220;cuff model&#8221; of neuropathic pain in mice consists of the implantation of a polyethylene cuff around the main branch of the sciatic nerve. Mechanical allodynia is tested using von Frey filaments.

Neuropathic pain arises as a consequence of a lesion or a disease affecting the somatosensory system. This syndrome results from maladaptive changes in ...

Monitoring of Systemic and Hepatic Hemodynamic Parameters in Mice

The use of mouse models in experimental research is of enormous importance for the study of hepatic physiology and pathophysiological disturbances. However, due to the small size of the mouse, technical details of the intraoperative monitoring procedure suitable for the mouse were rarely described. Previously we have reported a monitoring procedure to obtain hemodynamic parameters for rats. Now, we adapted the procedure to acquire systemic and hepatic hemodynamic parameters in mice, a species ten-fold smaller than rats. This film demonstrates the instrumentation of the animals as well as the data acquisition process needed to assess systemic and hepatic hemodynamics in mice. Vital parameters, including body temperature, respiratory&#160;rate&#160;and heart&#160;rate were recorded throughout the whole procedure. Systemic hemodynamic parameters consist of carotid artery pressure (CAP) and central venous pressure (CVP). Hepatic perfusion parameters include portal vein pressure (PVP), portal flow rate as well as the flow rate of the common hepatic artery (table 1). Instrumentation and data acquisition to record the normal values was completed within 1.5 h. Systemic and hepatic hemodynamic parameters remained within normal ranges during this procedure.
This procedure is challenging but feasible. We have already applied this procedure to assess hepatic hemodynamics in normal mice as well as during 70% partial hepatectomy and in liver lobe clamping experiments. Mean PVP after resection (n= 20), was 11.41&#177;2.94 cmH2O which was significantly higher (P&#60;0.05) than before resection (6.87&#177;2.39 cmH2O). The results of liver lobe clamping experiment indicated that this monitoring procedure is sensitive and suitable for detecting small changes in portal pressure and portal flow rate. In conclusion, this procedure is reliable in the hands of an experienced micro-surgeon but should be limited to experiments where mice are absolutely needed.

This film demonstrates how to acquire systemic and hepatic hemodynamics in mice. The whole monitoring includes acquisition of vital parameters, systemic blood pressure, central venous pressure, common hepatic artery flow rate, and portal vein pressure as well as the portal flow rate in mice.

The use of mouse models in experimental research is of enormous importance for the study of hepatic physiology and pathophysiological disturbances. ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

The Pilocarpine Model of Temporal Lobe Epilepsy and EEG Monitoring Using Radiotelemetry System in Mice

Temporal lobe epilepsy (TLE) is a common neurological disorder in adulthood. For translational studies of chronic epilepsy, pilocarpine-induced status epilepticus (SE) is frequently selected to recapitulate spontaneous recurrent seizures (SRS). Here we present a protocol of SE induction by intraperitoneal (i.p.) injection of pilocarpine and monitoring of chronic recurring seizures in live animals using a wireless telemetry video and electroencephalogram (EEG) system. We demonstrated notable behavioral changes that need attention after pilocarpine injection and their correlation with hippocampal neuronal loss at 7 days and 6 weeks post-pilocarpine. We also describe the experimental procedures of electrode implantation for video and EEG recording, and analysis of the frequency and duration of chronic recurrent seizures. Finally, we discuss the possible reasons why the expected results are not achieved in each case. This provides a basic overview of modeling chronic epilepsy in mice and guidelines for troubleshooting. We believe this protocol can serve as a baseline for suitable models of chronic epilepsy and epileptogenesis.

This manuscript describes a method of inducing status epilepticus by systemic pilocarpine injection and of monitoring spontaneous recurrent seizures in live animals using a wireless telemetry video and electroencephalogram system. This protocol can be utilized for studying the pathophysiologic mechanisms of chronic epilepsy, epileptogenesis, and acute seizures.

Temporal lobe epilepsy (TLE) is a common neurological disorder in adulthood. For translational studies of chronic epilepsy, pilocarpine-induced status ...

Murine Distal Colostomy, A Novel Model of Diversion Colitis in C57BL/6 Mice

Diversion colitis (DC) is a frequent clinical condition occurring in patients with bowel segments excluded from the fecal stream as a result of a diverting enterostomy. The etiology of this disease remains ill-defined but appears to differ from that of classical inflammatory bowel diseases such as Crohn's disease and ulcerative colitis. Research aimed to decipher the pathophysiological mechanisms leading to the development of this disease has been severely hampered by the lack of an appropriate murine model. This protocol generates a murine model of DC that facilitates the study of the immune system's role and its interaction with the microbiome in the development of DC. In this model using C57BL/6 animals, distal parts of the colon are excluded from the fecal stream by creating a distal colostomy, triggering the development of mild to moderate inflammation in the excluded bowel segments and reproducing the hallmark lesions of human DC with a moderate systemic inflammatory response. In contrast to the rat model, a large number of genetically-modified murine models on the C57BL/6 background are available. The combination of these animals with our model allows the potential roles of individual cytokines, chemokines, or receptors of bioactive molecules (e.g., interleukin (IL)-17; IL-10, chemokine CXCL13, chemokine receptors CXCR5 and CCR7, and the sphingosine-1-phosphate receptor 4) to be assessed in the pathogenesis of DC. The availability of congenic mouse strains on the C57BL/6 background largely facilitates transfer experiments to establish the roles of distinct cell types involved in the etiology of DC. Finally, the model offers the opportunity to assess the influences of local interventions (e.g., modification of the local microbiome or local anti-inflammatory therapy) on mucosal immunity in affected and non-affected bowel segments and the on systemic immune homeostasis.

Murine distal colostomy provides a murine model for human diversion colitis, a predominantly lymphocytic colitis in colon segment excluded from the fecal stream.

Diversion colitis (DC) is a frequent clinical condition occurring in patients with bowel segments excluded from the fecal stream as a result of a ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...