Name: isolation protocol of mouse monocyte-derived dendritic cells and their subsequent in vitro activation with tumor immune complexes
Uploaded: 2018-05-31T13:00:00
Description: Watch this Scientific Journal Video about Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes at JoVE.com

The protocols below refer to the isolation of mouse MoDC, yet the overall principles may apply to other DC subsets cells, as well. 12 - 16-week-old C57Bl/6j mice were maintained in an American Association for the Accreditation of Laboratory Animal Care&#8211;accredited animal facility. All protocols were approved by Stanford University and Tel-Aviv University Institutional Animal Care and Use Committee.
1. Isolation of Tumor Associated Monocyte-Derived DC
<ol>
	<li>In a laminar flow hood, re...

<ol>
	<li>Steinman, R. M., Banchereau, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Taking+dendritic+cells+into+medicine.">Taking dendritic cells into medicine.</a> Nature. 449 (7161), 419-426 (2007).</li><li>Palucka, K., Banchereau, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic-cell-based+therapeutic+cancer+vaccines.">Dendritic-cell-based therapeutic cancer vaccines.</a> Immunity. 39 (1), 38-48 (2013).</li><li>Mildner, A., Jung, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+function+of+dendritic+cell+subsets.">Development and function of dendritic cell subsets.</a> Immunity. 40 (5), 642-656 (2014).</li><li>Merad, M., Sathe, P., Helft, J., Miller, J., Mortha, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dendritic+cell+lineage:+ontogeny+and+function+of+dendritic+cells+and+their+subsets+in+the+steady+state+and+the+inflamed+setting.">The dendritic cell lineage: ontogeny and function of dendritic cells and their subsets in the steady state and the inflamed setting.</a> Annual review of immunology. 31, 563-604 (2013).</li><li>Guilliams, M., 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=Dendritic+cells,+monocytes+and+macrophages:+a+unified+nomenclature+based+on+ontogeny.">Dendritic cells, monocytes and macrophages: a unified nomenclature based on ontogeny.</a> Nature reviews. 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Given the large number of DC required for vaccinating mice (approximately 2-4 x 106 DC per one mouse), most of the vaccination strategies in mice are based on isolation of DC from BM and spleen followed by their ex vivo activation. However, attempts to activate tumor DC in vivo, using the same conditions for activating spleen and BM DC, have often been unsuccessful in producing effective immunity. In two subsequent publications, Carmi et al. have found that blood and tumor MoDC diffe...

Since their discovery, dendritic cells (DC) have been a focus of extensive research due to their unique ability to skew T cell differentiation1. Over the past several decades, an extensive research effort has sought to define the various DC subsets and their function during tumor progression and immunity 2. DCs are composed of heterogeneous cell populations that differ from each other in their pattern-recognition receptors, tissue distribution, and migratory and antigen presentation capabilities3,4,5. Compared to other DC subsets, monocyte-derived DC (MoDC) are far more abundant in tumors and can be easily generated from circulating or tumor-infiltrating monocytes6,7. Therefore, many clinical trials seeking to take advantage of their relative prevalence are based on in vivo and ex vivo manipulation of autologous MoDC in order to elicit T cell immunity 8,9.
Similarly, DC-based vaccination of experimental tumor models requires 2-3 serial injections, 5-7 days apart, of 1-2 x 106 activated DC pulsed with tumor antigens. Therefore, to achieve this large number of DC, most mouse studies have primarily used MoDC cultured from bone marrow (BM) precursors in GM-CSF for 7-9 days (IL-4 is not needed in the mouse setting)10,11. Nonetheless, given that GM-CSF knockout mice have overall normal DC compartment 12,13, and given the mixed populations obtained from that culture,14 the physiological relevance of these DC has been called into question.
Alternatively, DC may be routinely isolated from spleen cells. However, DC comprise only about 0.3-0.8% of total spleen cells (resulting in approximately 7 x 105 DC/spleen), and of these cells, only CD103+ DC and MoDC can migrate back to lymphoid organs. Since MoDCs comprise approximately 10-15% of splenic DC populations15,16, most isolation protocols yield approximately 1 x 105 MoDC per spleen. Expansion of MoDC can be achieved by injecting transfected B16 cells that secrete GM-CSF, resulting in a 100-fold increase in splenic MoDC17. However, the use of MoDC for developing DC vaccines is limited since this procedure cannot be done in humans and the obtained MoDC are already highly activated.
In addition to obtaining adequate numbers of DC, another challenge for developing effective DC vaccines against autologous cancer cells involves the lack of sufficient danger signals in the tumor setting to fully activate DC. Induction of co-stimulatory signals is usually achieved through activation of pattern-recognition receptors (PRR), or c-type lectin signaling pathways18,19,20,21. A further approach for activating DC exploits their ability to take up antigens through interactions with surface Fc&#947; receptors (Fc&#947;R). Indeed, a number of important manuscripts have shown that injection of MoDC from BM precursors activated with tumor-IgG IC can prevent tumor growth in prophylactic settings, and can lead to the eradication of established tumors22,23.
In two recent papers, Carmi et al. discovered that in contrast to BMDC and spleen DC, MoDC from the blood and tumors cannot respond to IgG IC without additional stimuli. This was found to be due to the presence of high intracellular levels of tyrosine phosphatases regulating Fc&#947;R signaling24,25. By defining a critical checkpoint in DC, this work provided an important insight into the requirements for successful DC-based vaccination. The requirement for additional stimuli to enable Fc&#947;R signaling, and presumably signaling from other lectin receptors utilizing a similar phosphorylation cascade, thus underscores the need for avoiding the priming of DC during their isolation.
Therefore, the present protocol describes the isolation of MoDC from blood and tumors, which differ markedly from BM and spleen DC, and highlights precautions worth considering during the process.

<table>
	<tbody>
		<tr>
			<td>Ficoll-Paque PREMIUM</td>
			<td>GE-Healthcare</td>
			<td>17-5442-02</td>
			<td></td>
		</tr>
		<tr>
			<td>OptiPrep</td>
			<td>StemCell Technologies</td>
			<td>07820</td>
			<td></td>
		</tr>
		<tr>
			<td>CD45 MicroBeads</td>
			<td>Miltenyi</td>
			<td>130-052-301</td>
			<td></td>
		</tr>
		<tr>
			<td>EasySep Monocyte Isolation Kit</td>
			<td>StemCell Technologies</td>
			<td>19861</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase IV</td>
			<td>Sigma</td>
			<td>C9697-50MG</td>
			<td>Test each lot for endotoxin</td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Sigma</td>
			<td>DN25-10MG</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>ThermoFisher</td>
			<td>14025092</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>ThermoFisher</td>
			<td>16140071</td>
			<td>Test each lot for endotoxin</td>
		</tr>
		<tr>
			<td>PE-CD11c</td>
			<td>Biolegend</td>
			<td>117307</td>
			<td></td>
		</tr>
		<tr>
			<td>APC-CD11b</td>
			<td>Biolegend</td>
			<td>101211</td>
			<td></td>
		</tr>
		<tr>
			<td>Brilliant Violet 650 MHCII</td>
			<td>Biolegend</td>
			<td>107641</td>
			<td></td>
		</tr>
		<tr>
			<td>AF48- CD86</td>
			<td>Biolegend</td>
			<td>105017</td>
			<td></td>
		</tr>
		<tr>
			<td>APC/Cy7-Ly-C6</td>
			<td>Biolegend</td>
			<td>108423</td>
			<td></td>
		</tr>
		<tr>
			<td>PE/Cy7-CD15</td>
			<td>Biolegend</td>
			<td>135523</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation protocol of mouse monocyte-derived dendritic cells and their subsequent in vitro activation with tumor immune complexes

Dendritic cells (DC) are heterogeneous cell populations that differ in their cell membrane markers, migration patterns and distribution, and in their antigen presentation and T cell activation capacities. Since most vaccinations of experimental tumor models require millions of DC, they are widely isolated from the bone marrow or spleen. However, these DC significantly differ from blood and tumor DC in their responses to immune complexes (IC), and presumably to other Syk-coupled lectin receptors. Importantly, given the sensitivity of DC to danger-associated molecules, the presence of endotoxins or antibodies that crosslink activation receptors in one of the isolating steps could result in the priming of DC and thus affect the parameters, or at least the dosage, required to activate them. Therefore, here we describe a detailed protocol for isolating MoDC from blood and tumors while avoiding their premature activation. In addition, a protocol is provided for MoDC activation with tumor IC, and their subsequent analyses.

We initially compared the capacity of antibodies from na&#239;ve syngeneic and allogeneic mice to bind to tumor cells. To this end, B16F10 and LMP tumor cell lines were fixed in paraformaldehyde and washed extensively. B16F10 is a melanoma cell line, which was originally isolated from lung metastases in C57Bl/6 mice. LMP is a pancreatic tumor cell that was isolated from KrasG12D/+, LSL-Trp53R172H/+, and Pdx-1-Cre mice, and grows steadily in 129F1 mice. To obtain IC, tumor cells were incub...

Watch this Scientific Journal Video about Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes at JoVE.com

Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes

Monocyte-derived DC (MoDC) can sense minor amounts of danger-associated molecules and are therefore easily primed. We provide a detailed protocol for the isolation of MoDC from blood and tumors and their activation with immune complexes while highlighting key precautions that should be considered in order to avoid their premature activation.

Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes

Dendritic cells (DC) are heterogeneous cell populations that differ in their cell membrane markers, migration patterns and distribution, and in their ...

This method is designed to help scientists to isolate monocyte-derived DCs from the blood and tumors of tumor-bearing mice in order to study their immunostimulatory properties. I think that the main advantage of this procedure compared to other method is that the obtained monocyte-derived DCs better reflect their activation state in the tumor and blood. The hardest thing is probably to make sure that all of your reagents and tools are sterile and that you will not introduce contamination during this procedure.I've been isolating myeloid cells and dendritic cells from tumors for over 15 years now, and I had the idea of this method when I noticed that different isolation protocols resulted in different patterns of cell activation. Demonstrating the procedure will be Mrs. Santana-Magal, who is a grad student in my lab.After euthanizing the mice using carbon dioxide inside a laminar flow hood, extract the tumors and place them in RPMI medium without fetal bovine serum. Then, use surgery scissors to slice the tumor into small pieces. Transfer all of the tumor pieces from a single mouse to a 30-milliliter flat-bottom tube containing HBSS buffer supplemented with collagenase IV and DNase I along with the magnetic stir bars.Incubate the tube with the tumor pieces on a shaker at 37 degrees Celsius with a magnetic stirrer inside at 200 to 400 revolutions per minute for 20 to 30 minutes. Next, filter the cells through a 70-micrometer cell strainer. Then, centrifuge the cells at 400 times g for five to 10 minutes at four degrees Celsius.After centrifugation, add 1.5 milliliters of density gradient medium stock solution to 8.5 milliliters of HBSS. Then, mix the density gradient medium vigorously. Next, pipette the mixture to the pellet.Centrifuge the cell suspension at 400 times g for 20 minutes at room temperature. Decant the supernatant after the centrifugation is over. After washing the pelleted cells twice, resuspend in one milliliter of isolation buffer.Add the cell suspension to 30 microliters of CD11b conjugated magnetic beads. Next, incubate the mixture at four degrees Celsius for 15 minutes. Following centrifugation, remove the supernatant and resuspend the pellet in one milliliter of isolation buffer.Apply the cell suspension on a pre-washed magnetic column. Next, use three milliliters of isolation buffer to wash the column twice. Then, remove the column from the magnet.Next, add six milliliters of isolation buffer in the column. Push with a plunger to flush out the magnetically labeled cells in a sterile collection tube. Once again, centrifuge the cells at 400 times g for five to 10 minutes at four degrees Celsius.After resuspending and staining the cells, sort the cells by gating the small cells, using small side and small forward scatter. After euthanizing the mouse, spray 70%ethanol on it. Then, use surgical scissors to remove the skin covering the heart under the laminar hood.Next, clean the scissors with ethanol. Wash the cavity with 20-millimolar EDTA HBSS, and use the scissors to cut the right atrium of the heart. Then, use a 10-milliliter syringe with a 25-gauge needle to slowly flush the right ventricle of the heart with 20-millimolar EDTA HBSS.Then, use a sterile syringe to draw blood from the pleural cavity. Transfer the blood in a tube containing heparan sulfate and EDTA. Next, pipette the blood on the density gradient medium.Centrifuge the blood cells at 400 times g for 15 minutes at room temperature with low brake. After centrifugation, collect the mononuclear cells in a tube. Dissolve the cells in 10 milliliters of isolation buffer to wash the cells.Again, centrifuge the cells at 400 times g for five to 10 minutes at four degrees Celsius. To select the CD11b cells, resuspend the cell pellet in one milliliter of isolation buffer. Incubate for 15 minutes with 50 microliters of CD11b conjugated magnetic beads at four degrees Celsius.Recentrifuge at 400 times g for five to 10 minutes at four degrees Celsius. Then, remove the supernatant. Resuspend the pellet in one milliliter of isolation buffer.Next, apply the cell suspension on a pre-washed magnetic column. Wash the column with isolation buffer twice. Remove the column from the magnet, and add six milliliters of isolation buffer on the column.Next, flush out the magnetically labeled cells in a sterile collection tube by pushing the plunger. Centrifuge the cells at 400 times g for five to 10 minutes at four degrees Celsius. Resuspend the cells in isolation buffer and stain with fluorophore-conjugated antibodies.Sort the cells by gating the small cells using small side and small forward scatter. First, fix the cells in 1.8%buffered paraformaldehyde for 10 minutes at room temperature. Seed the cell suspension in each well of a U-shaped 96-well plate.Next, add varying dilutions of tumor-binding antibodies in each well. Thereafter, incubate the cells on ice for 15 to 20 minutes. After incubation, wash the cells with 150 microliters of phosphate buffered saline.Then, centrifuge at 400 times g for five to 10 minutes at four degrees Celsius. Post-centrifugation, expel the supernatant and wash the cells twice. Dissolve the cells in 100 microliters of FACS buffer containing fluorophore-conjugated secondary antibody.Next, incubate the plate on ice for 15 to 20 minutes. After 15 to 20 minutes, add 200 microliters of FACS buffer to wash the cells. Centrifuge the plate at 400 times g for five to 10 minutes.Decant the supernatant. Conduct flow cytometry to analyze the tumor binding and to determine the minimal concentration of immunoglobulin G required to coat the cells. Once the cells are coated with minimal immunoglobulin G concentration, add the CFSE-labeled tumor immune complex to the monocyte-derived dendritic cells in a one-to-five ratio.Then, incubate the mixture for 12 to 16 hours overnight in complete medium. Perform FACS analysis of the monocyte-derived dendritic cell activation. Mean fluorescence intensity is calculated following flow cytometric analysis to study the presence of IgG and M antibodies that bind LMP tumor cells ex vivo.Results show that the IgG antibodies from C57-Black/6 allogeneic mice bind the tumor cells much stronger than the antibodies from the 129S1 syngeneic mice. Then, the mean fluorescence intensity of the binding capacity of B16F10 tumor cells with the IgG is calculated. IgG obtained from 129S1 allogeneic mice shows 10-fold higher staining intensity with B16F10 tumor in comparison to syngeneic IgG from C57-Black/6 mice.Next, DIC microscopic images are captured to show the tumor-associated, monocyte-derived dendritic cells from the B16F10 tumors. Here, DIC images are captured to show the inflammatory and patrolling monocyte-derived dendritic cells isolated from the blood of B16F10 tumor-bearing mice. Flow cytometric analysis is also done to compare the activation patterns of monocyte-derived dendritic cells from the spleen and the bone marrow with that of the tumor and blood on incubating with the immune complex.The result shows increased CD86 and MHC II expression by the bone marrow and splenic dendritic cells with the immune complexes. Then, confocal immunohistochemistry is done, which shows tumor uptake and MHC II expression when the dendritic cells are incubated with the CFSE-labeled tumor immune complex. Once mastered, this procedure can be done in a few hours depending on the number of mice you are using.While attempting this procedure, it's important to remember to keep all your reagents and tools sterile at all time. Mouse fur is a major source for contaminations. Make sure that none of the hairs are touching your tissues.We hope that after watching this video, you'll have a good understanding of how to isolate monocyte-derived DCs from mouse blood and tumors and how to subsequently activate them with immune complex cells while avoiding their premature activation.

Research

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