We thank Reata Pharmaceuticals for providing CDDO-DFPA. We also acknowledge the support of the Jane and Lee Seidman Chair in Pediatric Cancer Innovation (John Letterio). This work was supported by Department of Defense [W81XWH-12-1-0452]; the Angie Fowler Adolescent and Young Adult Cancer Research Initiative at the Case Comprehensive Cancer Center; and the Callahan Graduate Scholar Award for Hsi-Ju Wei from F.J. Callahan Foundation.

All studies were performed in compliance with procedures approved by the Case Western Reserve University School of Medicine&#39;s Institutional Animal Care and Use Committee.
1. Prepare Bone Marrow-derived Dendritic Cells (BMDCs)
<ol>
	<li>Sterilize all the surgical instruments via autoclaving and perform the experiment in class II biological safety cabinet with appropriated safety procedures.</li>
	<li>Euthanize C57BL/6 mice 8 - 10 weeks of age by using CO2 chamber. Place the mouse on a dissecting board and rinse with 70% ethanol. Excise tibia-fibula and femur bones by using a surgical scissor and place them with 70% ethanol in a 10 cm culture dish.</li>
	<li>Use surgical blades and forceps to dissect tissue away as much as possible on the lid of the 10 cm culture dish and isolate tibias and femurs to place them with 70% ethanol in a 6 cm culture dish.</li>
	<li>Use surgical blades to trim both ends of tibias and femurs. Use 3 mL PBS in 3 ml syringe with a 23G needle to flush the contents of marrow from one end of bones to a conical tube containing 12 ml PBS. Repeat this step 3x for each end of bones.</li>
	<li>Centrifuge the cell suspension at 300 x g for 5 min.</li>
	<li>Remove the supernatant and resuspend the cell pellet with 1 ml ACK lysing buffer for 5 mins to remove red blood cells.</li>
	<li>Add 9 ml PBS to dilute ACK lysing buffer and centrifuge at 300 x g for 5 mins.</li>
	<li>Remove the supernatant and resuspend with 10 ml culture medium (RPMI-1640 plus L-glutamine, 10% FBS, 1% non-essential amino acid (100x), 10 mM HEPES, 50 nM &#946;-mercaptoethanol, and 5% penicillin/streptomycin). 
	NOTE: Endotoxin level has to be less than 0.1 EU/ml in FBS.</li>
	<li>Pass the cell suspension through a 40 &#956;m cell strainer and take 20 &#181;l of cell suspension and mix with 80 &#181;l of trypan blue to count the number of live cells by using cytometer.</li>
	<li>Adjust the cell number to 1 x 106 cells/ml with 15 ng/ml GM-CSF and 10 ng/ml IL-4.</li>
	<li>Plate 3 ml of 1 x 106 cells/ml in each well of 6-well plate and incubate the cells at 37&#176;C, 5% CO2, and 95% humidity in the CO2 incubator. 
	NOTE: The bone marrow cells from one mouse is around 3 - 5 x 107 cells, indicating that it can be placed from 2 to 3 6-well plates.</li>
	<li>On day 3, remove all 3 ml culture medium from each well, add 2 ml fresh PBS in each well, and then gently swirl the plate to ensure removing all the non-adherent cells.</li>
	<li>Replace 3 ml fresh culture medium with 15 ng/ml GM-CSF and 10 ng/ml IL-4 in each well. Incubate the cells at 37 &#176;C, 5% CO2, and 95% humidity in the CO2 incubator.</li>
	<li>On day 5, directly add another 3 ml fresh culture medium with 15 ng/ml GM-CSF and 10 ng/ml IL-4 in each well. Incubate the cells at 37&#176;C, 5% CO2, and 95% humidity in the CO2 incubator. 
	NOTE: The total volume in each well is now 6 ml.</li>
	<li>On day 7, put the entire plate on ice for 10 mins. Gently pipette the culture medium in each well to dislodge the loosely-adherent BMDCs as iDCs into suspension. 
	NOTE: The adherent macrophages are still attached to the plate. Low temperature and gently procedures are the key to avoid DC activation to affect further experiments.</li>
	<li>Centrifuge the cell suspension at 300 x g for 5 mins and resuspend with fresh culture medium for further experiments. 
	NOTE: BMDCs can be identified with fluorescence-labeled CD11c antibody by flow cytometry and we showed our gating strategy of BMDCs for further experiments in Figure 1.</li>
</ol>
2. Characterize TolDC Gene and Protein Profile
<ol>
	<li>Plate 2 ml of 1 x 106 BMDCs/ml per well in 6 well-plate with culture medium in the presence or absence of 100-400 nM CDDO-DFPA for incubating 1 h at 37&#176;C, 5% CO2 and 95% humidity in the CO2 incubator. 
	NOTE: CDDO-DFPA, a synthetic triterpenoid, is a nuclear factor (erythroid-derived 2)-like-2 factor (Nrf2) inducer and nuclear factor-&#954;B (NF-&#954;B) inhibitor. Apply other agents for induction of TolDCs from iDCs, such as IL-10, vitamin D3, dexamethasone or BAY 11-7085, and modify this step to an optimal condition for each agent.</li>
	<li>Add 10 or 100 ng/ml of LPS for incubating 4-24 hrs to induce mDCs (incubation time is different due to mRNA or protein measurement).</li>
	<li>Gently pipette the culture medium in each well and harvest the cell suspension by using 1 ml pipette. Centrifuge at 300 x g for 5 mins to collect the cells and supernatant, respectively.</li>
	<li>Analyze the surface ligands from the cell pellets by flow cytometry, such as stimulatory ligands: CD40, CD80, CD86, MHC-II, OX40L, ICOSL, or inhibitory ligands: PD-L1, PD-L2, ILT3, ILT4.</li>
	<li>Isolate the RNA from the cell pellets and analyze the RNA and supernatant samples for the cytokine profile and gene and protein levels by quantitative real-time PCR (qRT-PCR) and ELISA, respectively. 
	NOTE: For example, inflammatory cytokines: TNF-&#945;, IFN&#947;, EDN-1, IL-6, IL-12, and IL-23, or anti-inflammatory cytokines: IL-4, IL-10, IL-15, TGF-&#946;1, and HO-1.</li>
</ol>
3. Evaluate the Function of TolDCs In Vitro and In Vivo
<ol>
	<li>T-cell Syngeneic Proliferation Assay
	<ol>
		<li>Sterilize all the surgical instruments via autoclaving and perform the experiment in class II biological safety cabinet with appropriated safety procedures.</li>
		<li>Prepare MACS buffer using 0.5% bovine serum albumin (BSA) and 2 mM EDTA in 500 ml PBS. Sterilize the buffer by filtering through a 0.2 &#181;m filter. 
		NOTE: Keep the buffer on the ice during the following experiment.</li>
		<li>To obtain CD4+ T cells, euthanize OT-II T-cell receptor (TCR) transgenic mice 8-10 weeks of age by using CO2 chamber. Place the mouse on a dissecting board and rinse with 70% ethanol. Isolate the spleen from the left side of the abdomen by using surgical scissors and forceps.</li>
		<li>Put the spleen with 2 ml PBS in a 6 cm culture dish and use the back of push-stick of 3 ml syringe to mince the spleen by passing a 40 &#956;m cell strainer.</li>
		<li>Collect the cell suspension and centrifuge at 300 x g for 5 mins.</li>
		<li>Resuspend the cell pellet in 400 &#956;l of MACS buffer.</li>
		<li>Add 100 &#956;l of CD4+ T Cell Biotin-Antibody Cocktail at 4&#176;C for 5 mins. 
		NOTE: The antibody cocktail binds on other cell types except CD4+ T cells, such as CD8a, CD11b, CD11c, CD19, CD25, CD45R (B220), CD49b (DX5), CD105, Anti-MHC class II, Ter-119, and TCR&#947;/&#948;.</li>
		<li>Add 300 &#956;l of MACS buffer and 200 &#956;l of Anti-Biotin beads (Table of Materials) at 4&#176;C for 10 mins.</li>
		<li>Place the column composed of ferromagnetic spheres (Table of Materials) and Pre-Separation Filter together in the magnetic field and rinse it with 3 ml of MACS buffer.</li>
		<li>Add 9 ml of MACS buffer in the cells and centrifuge at 300 x g for 5 mins.</li>
		<li>Resuspend the cell pellet in 3 ml of MACS buffer and apply onto the column. Collect flow-through containing CD4+ T cells.</li>
		<li>Wash column with another 3 ml of MACS buffer and also collect the flow-through.</li>
		<li>To obtain splenic pan DCs, euthanize C57BL/6 mice 8-10 weeks of age by using CO2 chamber. Place the mouse on a dissecting board and rinse with 70% ethanol. Isolate the spleen from the left side of the abdomen by using surgical scissors and forceps. Put the spleen in a 6cm culture dish containing 2 ml of collagenase D solution (2 mg/ml collagenase D dissolved in HBSS containing calcium, magnesium).</li>
		<li>Inject 1 ml of collagenase D solution to the spleen two times with a 1 ml syringe and a 25G needle. Cut the spleen into small pieces with small scissors.</li>
		<li>Shake and incubate at room temperature for 25 mins.</li>
		<li>Add 500 &#956;l of 0.5 M EDTA at room temperature for 5 mins. 
		NOTE: The steps from 3.1.13-3.1.14 are critical for increasing the yield of DCs.</li>
		<li>Now use the back of push-stick of 3 ml syringe to mince the spleen slurry by passing a 40 &#956;m cell strainer and collect the cell suspension by centrifuging at 300 x g for 5 mins.</li>
		<li>Resuspend the cell pellet in 350 &#956;l of MACS buffer, 50 &#956;l of FcR Blocking Reagent, and 100 &#956;l of Pan Dendritic Cell Biotin-Antibody Cocktail at 4&#176;C for 10 mins. 
		NOTE: The antibody cocktail against antigens that are not expressed by DCs.</li>
		<li>Wash the cells by adding 9 ml of MACS buffer and centrifuge at 300 x g for 5 mins.</li>
		<li>Resuspend the cell pellet in 800 &#956;l of MACS buffer and add 200 &#956;l of Anti-Biotin beads at 4&#176;C for 10 mins.</li>
		<li>Repeat the steps from 3.1.9-3.1.11 to collect DCs.</li>
		<li>Wash column with another 3 ml of MACS buffer two times and also collect the flow-through.</li>
		<li>Treat the 2 x 105 DCs/ml in the presence or absence of 100-400 nM CDDO-DFPA at 37&#176;C for 1 hr. Seperately, label the CD4+ T cells (1 x 107/ml) with 1 &#956;M CFSE at 37&#176;C for 15 mins, wash with PBS, and readjust the volume to get final concentration at 2 x 106 T cells/ml.</li>
		<li>Next, in a 96-well plate, co-culture 100 &#956;l of the treated dendritic cells with the same volume of CFSE-labeled CD4+ T cells, both collected from the above steps to get 1:10 ratio. Now add 100 ng/mL ovalbumin (OVA) peptide 323-329 per well and measure the CFSE intensity of the T cells by flow cytometry after 2-3 days of incubation. 
		NOTE: The cell numbers and ratio of DCs and T cells have been optimized from our previous work37.</li>
	</ol>
	</li>
	<li>Induce Passive EAE by Injecting BMDCs Pulsed with Myelin Oligodendrocyte Glycoprotein (MOG) (35-55)
	<ol>
		<li>Plate 2 ml of 1 x 106 BMDCs/ml per well in 6 well-plate with culture medium in the presence or absence of 100-400 nM CDDO-DFPA for incubating 1 hr.</li>
		<li>Add 10 ng/ml of LPS for incubating 24 hrs.</li>
		<li>Add 100 &#956;g/ml of MOG (35-55) for incubating 4 hrs.</li>
		<li>Harvest the cell suspension and centrifuge at 300 x g for 5 mins.</li>
		<li>Resuspend the cell pellet with PBS and count the cells.</li>
		<li>Subcutaneously inject 200 &#956;l of 2 x 106 cells to 8-10 weeks old female C57BL/6 mice (100 &#956;l into each hind leg).</li>
		<li>On the day of BMDC injection and 48 hrs. later, inject 200 ng of pertussis toxin (PTX) in each mouse.</li>
		<li>Repeat steps 3.2.6-3.2.7 once each week for 4 consecutive weeks.</li>
		<li>Evaluate the clinical symptoms of EAE daily by using standard criteria37 (0.5-limp tail end, 1-limp tail, 2-moderate hind limb weakness, 3-severe hind limb weakness, 4-complete hind limb paralysis, 5-quadriplegia or moribund state, 6-death).</li>
	</ol>
	</li>
</ol>

This paper describes an efficient protocol that may be used to reproducibly to generate iDCs and to subsequently differentiate them into TolDCs, and we propose that this may be applied to evaluate the capacity of new molecular target agents to induce the TolDC phenotype. As described in this report, we followed a sequence in which we first analyzed TolDC expression of surface ligands by flow cytometry, followed by an assessment of the DC cytokine profile as measured by qRT-PCR and ELISA. Finally, the immunoregulatory fun...

Dendritic cells (DCs) are an integral part of the innate immune system and were first discovered and characterized by Ralph Steinman and Zanvil Cohn in 1973 as primary professional antigen presenting cells1. DCs have been shown to play an important role in immune activation by presenting processed antigens to T cells and B cells via major histocompatibility complexes (MHC) in secondary lymphoid organs to link the innate and adaptive immune systems2. In the mammalian immune system, there are at least two categories of DCs which have been described as myeloid DCs and plasmacytoid DCs (pDCs)3. Myeloid DCs, also known as conventional DCs (cDCs), are characterized by the expression of CD11c and can be differentiated as immature DCs (iDCs) in vitro from bone marrow progenitor cells or peripheral blood monocytes using granulocyte-macrophage-colony-stimulating factor (GM-CSF) and IL-4 in murine or human species, respectively4.
Activating &#39;danger&#39; signals, such as pathogen-associated molecular patterns (PAMP) or damage-associated molecular patterns (DAMP), will drive iDCs maturation toward immunogenic DCs as mature DCs (mDCs) via binding various pattern recognition receptors on the DC surface5. Immunogenic DCs further prime na&#239;ve T cell proliferation and differentiation through upregulation of MHCII2, costimulatory ligands (CD80, CD86, and CD40)6, cytokines, and other soluble mediators7. A cascade of pro-inflammatory mediator production from Immunogenic DCs is essential for cytokine-mediated T cell differentiation. For example, both IFN-&#947; and IL-12 are necessary for Th1 differentiation8 and IL-1, IL-6, and IL-23 are critical for na&#239;ve T cell polarization towards Th17 cells9. Although mature DCs react to foreign antigens, uncontrolled DC activation by self-antigens may cause tolerance ablation and foster the development of autoimmune diseases by generating autoreactive T cells whose activation leads to tissue destruction10.
Recent reports have provided clear evidence of DC plasticity, exemplified by their ability to interact with different cues within their tissue microenvironment and to differentiate into distinct effector/suppressor DC subsets. The anti-inflammatory mediators, such as IL-1011, TGF-&#946;12, and HO-113 have been shown to play an important role in immune suppression by inducing tolerogenic DCs (TolDCs). These TolDCs acquire regulatory functions and suppress T cell proliferation14. Moreover, the lack of co-stimulation by DCs and the production of anti-inflammatory mediators from TolDCs both contribute to the induction of regulatory T cells (Tregs) and also effectively inhibit both Th1 and Th17 differentiation and expansion15. In past two decades, the therapeutic potential of TolDCs has been reported by several investigators. In these studies, the administration of ex-vivo generated TolDCs not only ameliorated pathological symptoms in different preclinical models of autoimmune diseases16 but also led to the development of immune tolerance in patients17,18. Interestingly, today the TolDCs therapy has been considered as an alternative or adjunctive approach for autoimmune diseases in several clinical trials, including type 1 diabetes mellitus19, rheumatoid arthritis20,21, multiple sclerosis (MS)22,23,24, and Crohn&#39;s disease25.
There are a variety of protocols that have been employed to develop TolDCs and several laboratories have reported methods for generation and phenotypic characterization of TolDCs. These methods can be used to reproducibly generate TolDCs in vitro from hematopoietic progenitors and to stably maintain them in a tolerogenic state in vivo26,27,28,29. The iDCs can be converted into TolDCs by exposure to various immunomodulatory pharmacological agents or anti-inflammatory cytokines. For example, Vitamin D3 is a well-known pharmacological agent known to augment IL-10 production and suppress IL-12 secretion from DCs and to thereby boost their immunosuppressive function30. Moreover, when DCs are exposed to potent inflammatory stimuli, such as lipopolysaccharides (LPS), several pharmacological agents such as dexamethasone31, rapamycin32, and corticosteroids33 have been shown to induce the TolDC phenotype by reducing DC surface expression of CD40, CD80, CD86, and MHCII34. IL-10 and TGF-&#946; are the most-studied anti-inflammatory cytokines to induce DC tolerance35 and the concomitant exposure to both of these cytokines have been shown to induce a tolerogenic phenotype in DCs36.
Since the tolerogenic DC is defined by functional characteristics rather than by phenotypic markers, there is a great need to develop a consistent method for cellular and functional characterization of TolDCs. Moreover, a rigorous and consistent protocol must be established for the consistent evaluation and characterization of the tolerogenic DC phenotype if we are to effectively and reproducibly compare the ability of new agents to induce the TolDC phenotype in the laboratory. Here we provide a detailed protocol with step-by-step methods to isolate iDCs from hematopoietic progenitors of mice and to subsequently analyze the efficacy of new agents under evaluation for their capacity to convert iDCs into TolDCs, providing a robust functional and phenotypic characterization of TolDCs both in vitro and in vivo. This description includes an elaborate method to characterize the TolDCs by their surface ligands, cytokine profile, and immunosuppressive functions in vitro. We also provide an example of a method to explore the potential therapeutic application of these TolDCs in a pre-clinical model of MS, experimental autoimmune encephalomyelitis (EAE). This established protocol will help investigators to evaluate the capacity of new agents to promote the induction of TolDCs and will facilitate the effort to broaden the scope of TolDC therapeutic development.

<table>
	<tbody>
		<tr>
			<td>CDDO-DFPA (RTA-408)</td>
			<td>Reata Pharmaceuticals</td>
			<td>in house synthesis</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Mouse GM-CSF</td>
			<td>Peprotech Inc.</td>
			<td>315-03</td>
			<td>BMDC differentiation</td>
		</tr>
		<tr>
			<td>Mouse IL-4</td>
			<td>Peprotech Inc.</td>
			<td>214-14</td>
			<td>BMDC differentiation</td>
		</tr>
		<tr>
			<td>Lipopolysaccharides (LPS)</td>
			<td>Sigma Aldrich Inc.</td>
			<td>L2880</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>&#946;-mercaptoethanol</td>
			<td>Sigma Aldrich Inc.</td>
			<td>516732</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Pertussis toxin (PTX)</td>
			<td>R&#38;D systems</td>
			<td>3097</td>
			<td>EAE induction</td>
		</tr>
		<tr>
			<td>MOG (35&#8211;55) peptide</td>
			<td>21stCentury Biochemicals</td>
			<td>in house synthesis</td>
			<td>EAE induction</td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>Gibco, Life Technologies</td>
			<td>15250-061</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>RPMI-1640 plus L-glutamine</td>
			<td>ThermoFisher Scientific</td>
			<td>11875-093</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Non-essential amino acid (100X)</td>
			<td>ThermoFisher Scientific</td>
			<td>11140050</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>ThermoFisher Scientific</td>
			<td>15630080</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>penicillin/streptomycin</td>
			<td>ThermoFisher Scientific</td>
			<td>15140122</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>40 &#956;m cell strainer</td>
			<td>Corning</td>
			<td>352340</td>
			<td>Cell isolation</td>
		</tr>
		<tr>
			<td>PE-conjugated CD80</td>
			<td>BD Biosciences</td>
			<td>557227</td>
			<td>Flow cytometry</td>
		</tr>
		<tr>
			<td>PE-conjugated CD86</td>
			<td>BD Biosciences</td>
			<td>555665</td>
			<td>Flow cytometry</td>
		</tr>
		<tr>
			<td>PE-conjugated PD-L1</td>
			<td>BioLegend</td>
			<td>124307</td>
			<td>Flow cytometry</td>
		</tr>
		<tr>
			<td>APC-conjugated MHCII</td>
			<td>Miltenyi Biotec Inc.</td>
			<td>130-112-388</td>
			<td>Flow cytometry</td>
		</tr>
		<tr>
			<td>APC-conjugated CD11c</td>
			<td>BD Biosciences</td>
			<td>340544</td>
			<td>Flow cytometry</td>
		</tr>
		<tr>
			<td>Isotype matched PE</td>
			<td>Miltenyi Biotec Inc.</td>
			<td>130-091-835</td>
			<td>Flow cytometry</td>
		</tr>
		<tr>
			<td>Isotype matched APC</td>
			<td>Miltenyi Biotec Inc.</td>
			<td>130-091-836</td>
			<td>Flow cytometry</td>
		</tr>
		<tr>
			<td>CFSE</td>
			<td>BioLegend</td>
			<td>423801</td>
			<td>T cell proliferation assay</td>
		</tr>
		<tr>
			<td>Pan dendritic cell isolation kit</td>
			<td>Miltenyi Biotec Inc.</td>
			<td>130-100-875</td>
			<td>T cell proliferation assay</td>
		</tr>
		<tr>
			<td>FcR Blocking Reagent</td>
			<td>Miltenyi Biotec Inc.</td>
			<td>130-100-875</td>
			<td>T cell proliferation assay</td>
		</tr>
		<tr>
			<td>Pan Dendritic Cell Biotin-Antibody Cocktail</td>
			<td>Miltenyi Biotec Inc.</td>
			<td>130-100-875</td>
			<td>T cell proliferation assay</td>
		</tr>
		<tr>
			<td>Anti-Biotin MicroBeads</td>
			<td>Miltenyi Biotec Inc.</td>
			<td>130-100-875</td>
			<td>T cell proliferation assay</td>
		</tr>
		<tr>
			<td>CD4+ T cell isolation kit</td>
			<td>Miltenyi Biotec Inc.</td>
			<td>130-104-454</td>
			<td>T cell proliferation assay</td>
		</tr>
		<tr>
			<td>CD4+ T cell Biotin-Antibody Cocktail</td>
			<td>Miltenyi Biotec Inc.</td>
			<td>130-104-454</td>
			<td>T cell proliferation assay</td>
		</tr>
		<tr>
			<td>Anti-Biotin MicroBeads</td>
			<td>Miltenyi Biotec Inc.</td>
			<td>130-104-454</td>
			<td>T cell proliferation assay</td>
		</tr>
		<tr>
			<td>ACK lysing buffer</td>
			<td>ThermoFisher Scientific</td>
			<td>A1049201</td>
			<td>BMDC differentiation</td>
		</tr>
		<tr>
			<td>1 ml syringe</td>
			<td>BD Biosciences</td>
			<td>309626</td>
			<td>T cell proliferation assay</td>
		</tr>
		<tr>
			<td>3 ml syringe</td>
			<td>BD Biosciences</td>
			<td>309588</td>
			<td>BMDC differentiation</td>
		</tr>
		<tr>
			<td>25G needle</td>
			<td>BD Biosciences</td>
			<td>309626</td>
			<td>T cell proliferation assay</td>
		</tr>
		<tr>
			<td>23G needle</td>
			<td>BD Biosciences</td>
			<td>309588</td>
			<td>BMDC differentiation</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma Aldrich Inc.</td>
			<td>A2058</td>
			<td>T cell proliferation assay</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>ThermoFisher Scientific</td>
			<td>15575020</td>
			<td>T cell proliferation assay</td>
		</tr>
		<tr>
			<td>LS Column</td>
			<td>Miltenyi Biotec Inc.</td>
			<td>130-042-401</td>
			<td>T cell proliferation assay</td>
		</tr>
		<tr>
			<td>Pre-Separation Filter</td>
			<td>Miltenyi Biotec Inc.</td>
			<td>130-095-823</td>
			<td>T cell proliferation assay</td>
		</tr>
		<tr>
			<td>collagenase D</td>
			<td>Sigma Aldrich Inc.</td>
			<td>11088858001</td>
			<td>T cell proliferation assay</td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>ThermoFisher Scientific</td>
			<td>14025076</td>
			<td>T cell proliferation assay</td>
		</tr>
		<tr>
			<td>ovalbumin (OVA) peptide 323&#8211;329</td>
			<td>Sigma Aldrich Inc.</td>
			<td>O1641</td>
			<td>T cell proliferation assay</td>
		</tr>
		<tr>
			<td>Mouse IFN-&#947; TaqMan probe</td>
			<td>ThermoFisher Scientific</td>
			<td>Mm01168134_m1</td>
			<td>qRT-PCR</td>
		</tr>
		<tr>
			<td>Mouse IL-12a TaqMan probe</td>
			<td>ThermoFisher Scientific</td>
			<td>Mm00434165</td>
			<td>qRT-PCR</td>
		</tr>
		<tr>
			<td>Mouse IL-12 p70 DuoSet ELISA</td>
			<td>R&#38;D systems</td>
			<td>DY419-05</td>
			<td>ELISA</td>
		</tr>
		<tr>
			<td>Mouse EDN-1 ELISA</td>
			<td>RayBiotech</td>
			<td>ELM-EDN1-1</td>
			<td>ELISA</td>
		</tr>
		<tr>
			<td>TNF-&#945; TaqMan probe</td>
			<td>ThermoFisher Scientific</td>
			<td>Mm00443258</td>
			<td>qRT-PCR</td>
		</tr>
		<tr>
			<td>Mouse TNF-&#945; Quantikine ELISA Kit</td>
			<td>R&#38;D systems</td>
			<td>MTA00B</td>
			<td>ELISA</td>
		</tr>
		<tr>
			<td>IL-6 TaqMan probe</td>
			<td>ThermoFisher Scientific</td>
			<td>Mm00446190</td>
			<td>qRT-PCR</td>
		</tr>
		<tr>
			<td>Mouse IL-6 Quantikine ELISA Kit</td>
			<td>R&#38;D systems</td>
			<td>M6000B</td>
			<td>ELISA</td>
		</tr>
		<tr>
			<td>IL-23a TaqMan probe</td>
			<td>ThermoFisher Scientific</td>
			<td>Mm01160011</td>
			<td>qRT-PCR</td>
		</tr>
		<tr>
			<td>Mouse IL-23 DuoSet ELISA</td>
			<td>R&#38;D systems</td>
			<td>DY1887-05</td>
			<td>ELISA</td>
		</tr>
		<tr>
			<td>IL-4 TaqMan probe</td>
			<td>ThermoFisher Scientific</td>
			<td>Mm99999154_m1</td>
			<td>qRT-PCR</td>
		</tr>
		<tr>
			<td>IL-10 TaqMan probe</td>
			<td>ThermoFisher Scientific</td>
			<td>Mm01288386_m1</td>
			<td>qRT-PCR</td>
		</tr>
		<tr>
			<td>TGF-&#946; TaqMan probe</td>
			<td>ThermoFisher Scientific</td>
			<td>Mm01178820_m1</td>
			<td>qRT-PCR</td>
		</tr>
		<tr>
			<td>Anti-Heme Oxygenase 1 antibody</td>
			<td>Abcam</td>
			<td>ab13248</td>
			<td>Western blotting</td>
		</tr>
		<tr>
			<td>Anti-&#946;-actin antibody</td>
			<td>Abcam</td>
			<td>ab8226</td>
			<td>Western blotting</td>
		</tr>
		<tr>
			<td>CFX96 Touch Real-Time PCR Detection System</td>
			<td>Bio-Rad Inc.</td>
			<td></td>
			<td>qRT-PCR</td>
		</tr>
		<tr>
			<td>BD FACSCalibur Cell Analyzer</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>Flow cytometry</td>
		</tr>
	</tbody>
</table>

development and functional characterization of murine tolerogenic dendritic cells

The immune system operates by maintaining a tight balance between coordinating responses against foreign antigens and maintaining an unresponsive state against self-antigens as well as antigens derived from commensal organisms. The disruption of this immune homeostasis can lead to chronic inflammation and to the development of autoimmunity. Dendritic cells (DCs) are the professional antigen-presenting cells of the innate immune system involved in activating na&#239;ve T cells to initiate immune responses against foreign antigens. However, DCs can also be differentiated into TolDCs that act to maintain and promote T cell tolerance and to suppress effector cells contributing to the development of either autoimmune or chronic inflammation conditions. The recent advancement in our understanding of TolDCs suggests that DC tolerance can be achieved by modulating their differentiation conditions. This phenomenon has led to tremendous growth in developing TolDC therapies for numerous immune disorders caused due to break in immune tolerance. Successful studies in preclinical autoimmunity murine models have further validated the immunotherapeutic utility of TolDCs in the treatment of autoimmune disorders. Today, TolDCs have become a promising immunotherapeutic tool in the clinic for reinstating immune tolerance in various immune disorders by targeting pathogenic autoimmune responses while leaving protective immunity intact. Although an array of strategies has been proposed by multiple labs to induce TolDCs, there is no consistency in characterizing the cellular and functional phenotype of these cells. This protocol provides a step-by-step guide for the development of bone marrow-derived DCs in large numbers, a unique method used to differentiate them into TolDCs with a synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid-difluoro-propyl-amide (CDDO-DFPA), and the techniques used to confirm their phenotype, including analyses of essential molecular signatures of TolDCs. Finally, we show a method to assess TolDC function by testing their immunosuppressive response in vitro and in vivo in a preclinical model of multiple sclerosis.

The differentiation and selection of BMDCs: 
Bone marrow progenitor cells were cultured in complete RPMI medium in the presence of GM-CSF and IL-4 to differentiate into iDCs for 7 days (Figure 1A). On day 1, cells were small in size and showed spherical morphology. Washing with PBS before the replacement of fresh medium on Day 3 helped cells to form clusters and also increased the p...

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Development and Functional Characterization of Murine Tolerogenic Dendritic Cells

Here, we present a protocol to develop and characterize tolerogenic dendritic cells (TolDCs) and evaluate their immunotherapeutic utility.

The immune system operates by maintaining a tight balance between coordinating responses against foreign antigens and maintaining an unresponsive state ...

development-functional-characterization-murine-tolerogenic-dendritic

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10.5K Views.  Case Western Reserve University. Here, we present a protocol to develop and characterize tolerogenic dendritic cells (TolDCs) and evaluate their immunotherapeutic utility.

Video: Development and Functional Characterization of Murine Tolerogenic Dendritic Cells

Segmentation and Measurement of Fat Volumes in Murine Obesity Models Using X-ray Computed Tomography

Obesity is associated with increased morbidity and mortality as well as reduced metrics in quality of life.1 Both environmental and genetic factors are associated with obesity, though the precise underlying mechanisms that contribute to the disease are currently being delineated.2,3 Several small animal models of obesity have been developed and are employed in a variety of studies.4 A critical component to these experiments involves the collection of regional and/or total animal fat content data under varied conditions.
Traditional experimental methods available for measuring fat content in small animal models of obesity include invasive (e.g. ex vivo measurement of fat deposits) and non-invasive (e.g. Dual Energy X-ray Absorptiometry (DEXA), or Magnetic Resonance (MR)) protocols, each of which presents relative trade-offs. Current invasive methods for measuring fat content may provide details for organ and region specific fat distribution, but sacrificing the subjects will preclude longitudinal assessments. Conversely, current non-invasive strategies provide limited details for organ and region specific fat distribution, but enable valuable longitudinal assessment. With the advent of dedicated small animal X-ray computed tomography (CT) systems and customized analytical procedures, both organ and region specific analysis of fat distribution and longitudinal profiling may be possible. Recent reports have validated the use of CT for in vivo longitudinal imaging of adiposity in living mice.5,6 Here we provide a modified method that allows for fat/total volume measurement, analysis and visualization utilizing the Carestream Molecular Imaging Albira CT system in conjunction with PMOD and Volview software packages.

Fat content analysis is routinely conducted in studies utilizing murine obesity models. Emerging methods in small animal CT imaging and analysis are providing for longitudinal detail rich fat content analysis. Here we detail step by step procedures for performing small animal CT imaging, analysis, and visualization.

Obesity is associated with increased morbidity and mortality as well as reduced metrics in quality of life.1 Both environmental and genetic factors are ...

Development of Obliterative Bronchiolitis in a Murine Model of Orthotopic Lung Transplantation

Orthotopic lung transplantation in rats was first reported by Asimacopoulos and colleagues in 1971 1. Currently, this method is well accepted and standardized not only for the study of allo-rejection but also between syngeneic strains for examining mechanisms of ischemia-reperfusion injury after lung transplantation. Although the application of the rat and other large animal model 2 contributed significantly to the elucidation of these studies, the scope of those investigations is limited by the scarcity of knockout and transgenic rats. Due to no effective therapies for obliterative bronchiolitis, the leading cause of death in lung transplant patients, there has been an intensive search for pre-clinical models that replicate obliterative bronchiolitis. The tracheal allograft model is the most widely used and may reproduce some of the histopathologic features of obliterative bronchiolitis 3. However, the lack of an intact vasculature with no connection to the recipient's conducting airways, and incomplete pathologic features of obliterative bronchiolitis limit the utility of this model 4. Unlike transplantation of other solid organs, vascularized mouse lung transplants have only recently been reported by Okazaki and colleagues for the first time in 2007 5. Applying the basic principles of the rat lung transplant, our lab initiated the obliterative bronchiolitis model using minor histoincompatible antigen murine orthotopic single-left lung transplants which allows the further study of obliterative bronchiolitis immunopathogenesis6.

Obliterative bronchiolitis is the key impediment to the long-term survival of lung transplant recipients and the lack of a robust preclinical model precludes examining obliterative bronchiolitis immunopathogenesis. Unlike other solid organ transplants, vascularized mouse lung transplantation has only recently been developed. Here we show our independently developed obliterative bronchiolitis model after murine orthotopic single-lung transplantation.

Orthotopic lung transplantation in rats was first reported by Asimacopoulos and colleagues in 1971 1. Currently, this method is well accepted and ...

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Quantitative Analysis and Characterization of Atherosclerotic Lesions in the Murine Aortic Sinus

Atherosclerosis is a disease of the large arteries and a major underlying cause of myocardial infarction and stroke. Several different mouse models have been developed to facilitate the study of the molecular and cellular pathophysiology of this disease. In this manuscript we describe specific techniques for the quantification and characterization of atherosclerotic lesions in the murine aortic sinus and ascending aorta. The advantage of this procedure is that it provides an accurate measurement of the cross-sectional area and total volume of the lesion, which can be used to compare atherosclerotic progression across different treatment groups. This is possible through the use of the valve leaflets as an anatomical landmark, together with careful adjustment of the sectioning angle. We also describe basic staining methods that can be used to begin to characterize atherosclerotic progression. These can be further modified to investigate antigens of specific interest to the researcher. The described techniques are generally applicable to a wide variety of existing and newly created dietary and genetically-induced models of atherogenesis.

We describe procedures to quantify and characterize atherosclerotic lesions in mouse models using precision sectioning of the aortic sinus and ascending aorta combined with histochemical and immunohistochemical analysis.

Atherosclerosis is a disease of the large arteries and a major underlying cause of myocardial infarction and stroke. Several different mouse models have ...

Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and membrane ion currents. Those changes are likely&#160;to be responsible for the increased arrhythmogenic risk and the contractile alterations leading to systolic and diastolic dysfunction in cardiac patients. However, most information on the alterations of myocyte function in cardiac diseases has come from animal models.
Here we describe and validate a protocol to isolate viable myocytes from small surgical samples of ventricular myocardium from patients undergoing cardiac surgery operations. The protocol is described in detail. Electrophysiological and intracellular calcium measurements are reported to demonstrate the feasibility of a number of single cell measurements in human ventricular cardiomyocytes obtained with this method.
The protocol reported here can be useful for future investigations of the cellular and molecular basis of functional alterations of the human heart in the presence of different cardiac diseases. Further, this method can be used to identify novel therapeutic targets at cellular level and to test the effectiveness of new compounds on human cardiomyocytes, with direct translational value.

Current knowledge on the cellular basis of cardiac diseases mostly relies on studies on animal models. Here we describe and validate a novel method to obtain single viable cardiomyocytes from small surgical samples of human ventricular myocardium. Human ventricular myocytes can be used for electrophysiological studies and drug testing.

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and ...

Assessment of Ovarian Cancer Spheroid Attachment and Invasion of Mesothelial Cells in Real Time

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not accurately model the behaviors of cancer cells within a nonadherent environment, as cancer cells inherently aggregate into multicellular structures which contribute to the metastatic process by attaching to and invading the peritoneal lining to form secondary tumors. To model this important stage of ovarian cancer metastasis, multicellular aggregates, or spheroids, can be generated from established ovarian cancer cell lines maintained under nonadherent conditions. To mimic the peritoneal microenvironment encountered by tumor cells in vivo, a spheroid-mesothelial co-culture model was established in which preformed spheroids are plated on top of a human mesothelial cell monolayer, formed over an extracellular matrix barrier. Methods were then developed using a real-time cell analyzer to conduct quantitative real time measurements of the invasive capacity of different ovarian cancer cell lines grown as spheroids. This approach allows for the continuous measurement of invasion over long periods of time, which has several advantages over traditional endpoint assays and more laborious real time microscopy image analyses. In short, this method enables a rapid, determination of factors which regulate the interactions between ovarian cancer spheroid cells invading through mesothelial and matrix barriers over time.

Ovarian cancer cell invasion into the mesothelial lining of the peritoneum is a dynamic process over time. Utilizing a real time analyzer, the invasive capacity of ovarian cancer cells in a spheroid-mesothelial cell co-culture model can be quantified over prolonged time periods, providing insights into factors regulating the metastatic process.

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not ...

High Throughput Characterization of Adult Stem Cells Engineered for Delivery of Therapeutic Factors for Neuroprotective Strategies

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to develop strategies for treating a variety of diseases. The purpose of this study was to develop and characterize MSCs as cellular vehicles engineered for delivery of therapeutic factors as part of a neuroprotective strategy for rescuing the damaged or diseased nervous system. In this study we used mouse MSCs that were genetically modified using lentiviral vectors, which encoded brain-derived neurotrophic factor (BDNF) or glial cell-derived neurotrophic factor (GDNF), together with green fluorescent protein (GFP). 
Before proceeding with in vivo transplant studies it was important to characterize the engineered cells to determine whether or not the genetic modification altered aspects of normal cell behavior. Different culture substrates were examined for their ability to support cell adhesion, proliferation, survival, and cell migration of the four subpopulations of engineered MSCs. High content screening (HCS) was conducted and image analysis performed. 
Substrates examined included: poly-L-lysine, fibronectin, collagen type I, laminin, entactin-collagen IV-laminin (ECL). Ki67 immunolabeling was used to investigate cell proliferation and Propidium Iodide staining was used to investigate cell viability. Time-lapse imaging was conducted using a transmitted light/environmental chamber system on the high content screening system.
Our results demonstrated that the different subpopulations of the genetically modified MSCs displayed similar behaviors that were in general comparable to that of the original, non-modified MSCs. The influence of different culture substrates on cell growth and cell migration was not dramatically different between groups comparing the different MSC subtypes, as well as culture substrates. 
This study provides an experimental strategy to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair.

This study describes an experimental platform to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair.

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to ...

In vitro Functional Characterization of Mouse Colorectal Afferent Endings

This video demonstrates in detail an in vitro single-fiber electrophysiological recording protocol using a mouse colorectum-nerve preparation. The approach allows unbiased identification and functional characterization of individual colorectal afferents. Extracellular recordings of propagated action potentials (APs) that originate from one or a few afferent (i.e., single-fiber) receptive fields (RFs) in the colorectum are made from teased nerve fiber fascicles. The colorectum is removed with either the pelvic (PN) or lumbar splanchnic (LSN) nerve attached and opened longitudinally. The tissue is placed in a recording chamber, pinned flat and perfused with oxygenated Krebs solution. Focal electrical stimulation is used to locate the colorectal afferent endings, which are further tested by three distinct mechanical stimuli (blunt probing, mucosal stroking and circumferential stretch) to functionally categorize the afferents into five mechanosensitive classes. Endings responding to none of these mechanical stimuli are categorized as mechanically-insensitive afferents (MIAs). Both mechanosensitive and MIAs can be assessed for sensitization (i.e., enhanced response, reduced threshold, and/or acquisition of mechanosensitivity) by localized exposure of RFs to chemicals (e.g., inflammatory soup (IS), capsaicin, adenosine triphosphate (ATP)). We describe the equipment and colorectum&#8211;nerve recording preparation, harvest of colorectum with attached PN or LSN, identification of RFs in the colorectum, single-fiber recording from nerve fascicles, and localized application of chemicals to the RF. In addition, challenges of the preparation and application of standardized mechanical stimulation are also discussed.

In vitro Functional Characterization of Mouse Colorectal Afferent Endings

This video demonstrates a protocol for conducting single-fiber electrophysiological recordings on an in vitro mouse colorectum-nerve preparation.

This video demonstrates in detail an in vitro single-fiber electrophysiological recording protocol using a mouse colorectum-nerve preparation. The ...

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene and cell therapies are highly promising alternative approaches for CVD treatment. However, the broad clinical application of gene therapy is greatly limited by the lack of suitable gene delivery systems. The development of appropriate gene delivery vectors can provide a solution to current challenges in cell therapy. In particular, existing drawbacks, such as limited efficiency and low cell retention in the injured organ, could be overcome by appropriate cell engineering (i.e., genetic) prior to transplantation. The presented protocol describes the efficient and safe transient modification of endothelial cells using a polyethyleneimine superparamagnetic magnetic nanoparticle (PEI/MNP)-based delivery vector. Also, the algorithm and methods for cell characterization are defined. The successful intracellular delivery of microRNA (miR) into human umbilical vein endothelial cells (HUVECs) has been achieved without affecting cell viability, functionality, or intercellular communication. Moreover, this approach was proven to cause a strong functional effect in introduced exogenous miR. Importantly, the application of this MNP-based vector ensures cell magnetization, with accompanying possibilities of magnetic targeting and non-invasive MRI tracing. This may provide a basis for magnetically guided, genetically engineered cell therapeutics that can be monitored non-invasively with MRI.

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

This manuscript describes the efficient, non-viral delivery of miR to endothelial cells by a PEI/MNP vector and their magnetization. Thus, in addition to genetic modification, this approach allows for magnetic cell guidance and MRI detectability. The technique can be used to improve the characteristics of therapeutic cell products.

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene ...

Development and Functional Characterization of Murine Tolerogenic Dendritic Cells

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