This work was supported by NIH/NIAID grant AI45889 to S.A.P. Flow cytometry studies were carried out using FACS core facilities supported by the Einstein Cancer Center (NIH/NCI CA013330) and Center for AIDS Research (NIH/NIAID AI51519).

Animal experiments are done in accordance with approved guidelines from the institutional animal care and usage committee (IACUC). All procedures requiring sterility are performed in a biosafety cabinet.
1. Implantation of B16.Flt3L Melanoma in Mice
<ol>
	<li>Culture the Flt-3 expressing B16 melanoma cell line in T25 tissue culture flasks at a density of 0.5 x 106 cells/ml&#160;in complete DMEM medium with 10% CO2 at 37 &#176;C. Grow until the cells reach ~90% confluence (~20 hr).</li>
	<li>Harvest the cells by trypsin digestion. Aspirate cell culture media and wash the adherent cells with PBS. Add 5 ml&#160;of 0.05% trypsin and incubate for 5 min at 37 &#176;C. Add cold 20 ml&#160;complete DMEM medium (4 &#176;C) containing fetal calf serum (FCS) to quench the protease digestion. Lift the cells off by lightly tapping followed by gently pipetting up and down.</li>
	<li>Collect the cells by centrifugation at 300 x g for 10 min&#160;at 4 &#176;C. Count the cells using a hemocytometer or an automated cell viability counter. Resuspend to a density of 108 cells/ml&#160;in PBS.</li>
	<li>Implant mice (C57BL/6 or another histocompatible strain) with tumor cells by subcutaneous injection as described by Machholz et al.19 at the base of the neck with 100 &#181;l&#160;of cell suspension (107 cells).</li>
	<li>Allow the tumor to grow until palpable or visible (2 - 10 mm in diameter, usually 7 - 12 days) before sacrificing animals for harvesting organs.</li>
</ol>
2. Preparation of Splenic Single Cell Suspension from Tumor Bearing Mice
<ol>
	<li>Anesthetize mice with an overdose of isoflurane (adjust flow rate of isoflurane to &#62;5% and continue exposure at least 2 min&#160;after breathing stops). Sacrifice the Flt-3 melanoma tumor bearing mouse by cervical dislocation according to institutional guidelines for euthanasia.</li>
	<li>Disinfect the skin with 70% ethanol and harvest spleen using aseptic technique with the aid of sterile scissors 20.</li>
	<li>Place the spleen into a sterile Petri dish for transport to the biosafety cabinet. Perform all steps from here on under sterile conditions.</li>
	<li>Transfer spleen to a fresh Petri dish and wash with RPMI medium. Cut the spleen into small (~0.2 cm2) pieces using a scalpel. Incubate for 30 min at 37 &#176;C with 10 ml&#160;of collagenase/DNase solution.</li>
	<li>Pour the partially digested suspension of splenic tissue onto a 70 &#181;m cell strainer. Compress the remaining tissue fragments through the mesh of the strainer using a 5 ml&#160;syringe plunger.</li>
	<li>Wash the mesh filter with 10 ml&#160;of fresh medium and discard the filter. Centrifuge the suspension at 300 x g for 10 min&#160;at 4 &#176;C to pellet the cells.</li>
	<li>Aspirate the supernatant and resuspend the cell pellet in 2 ml&#160;of RBC lysis buffer. Incubate at RT for 10 min. Quench the RBC lysis buffer by adding 10 ml&#160;of complete RPMI medium.</li>
	<li>Pellet the cells by centrifugation at 300 x g for 5 min at 4 &#176;C. Resuspend in appropriate volume to achieve cell density of 108 cells/ml&#160;in cell sorting buffer (e.g., MACS) containing 20 &#181;g/ml&#160;of Fc-block antibody (2.4G2).</li>
</ol>
3. Purification of Plasmacytoid DCs, CD8&#945;Pos DCs and CD8&#945;Neg DCs from Splenic Single Cell Suspension
Note: Isolation of the splenic DC subsets from single cell suspension is a multi-step procedure as illustrated in Figure 1. Perform all the steps using pre-cooled buffer and an ice water bath to maintain temperature at 4 - 8 &#176;C.&#160;All reagent volumes in the following section of the protocol are calculated for an initial input of 200 &#181;l of splenic cell suspension at 108 cells/ml). This can be scaled up or down based on your need by changing the volume of cell suspension (always at a density of 1 X 108 cells/ml) and other reagents proportionately in the initial step (3.1.1 and 3.2.1). Adjust reagent volumes in all later steps accordingly. For example, if starting with 100 &#181;l of splenic cell suspension at 1 x 108/ml, use half the stated reagent volumes in all steps.
<ol>
	<li>Isolation of Plasmacytoid DCs (pDC)
	<ol>
		<li>Add 300 &#181;l&#160;of biotin-labeled antibody cocktail provided in the plasmacytoid DC isolation kit to 200 &#181;l&#160;of splenic cell suspension.</li>
		<li>Mix thoroughly and incubate in ice water bath for 15 min. Tap the tube every 3 min&#160;to keep cells suspended, and to maximize antibody binding.</li>
		<li>Add 12 ml&#160;cell sorting buffer and pellet the cells by centrifugation at 300 x g&#160;for 5 min at 4 &#176;C. Aspirate supernatant to remove unbound biotinylated antibodies.</li>
		<li>Resuspend the cell pellet in 500 &#181;l&#160;of cell sorting buffer. Add 300 &#181;l&#160;of anti-biotin antibody-conjugated beads. Repeat step 3.1.2 and 3.1.3.</li>
		<li>Discard supernatant and resuspend the cell pellet in 2 ml&#160;of cell sorting buffer. Perform all steps from this point on at RT&#160;using pre-cooled buffers.</li>
		<li>Place a fresh magnetic activated cell sorting LS column in the magnetic stand. Wash and equilibrate column with 5 ml&#160;of cell sorting buffer.</li>
		<li>Pipette the cell suspension onto the column, applying it gently to the center of the surface of the column bed. Collect the flow through.</li>
		<li>Wash the column with 10 ml&#160;of cell sorting buffer. Collect this flow through and combine with the flow through from step 3.1.7. The pDCs are enriched in this column flow through (FT1).</li>
		<li>Pellet the cells from FT1 by centrifugation at 300 x g for 5 min at 4 &#176;C, resuspend in 2 ml&#160;cell sorting buffer and pass the cells through a fresh LS column by repeating steps 3.1.6 - 3.1.8. This use of two sequential columns yields enhanced purity of isolated cells.</li>
		<li>Pellet cells by centrifugation at 300 x g for 5 min at 4 &#176;C, and resuspend with 2 ml&#160;complete RPMI. Place on ice until needed.</li>
	</ol>
	</li>
	<li>Isolation of CD8&#945;Pos and CD8&#945;Neg DCs 
	Note: The first step in this procedure is depletion of B, pDC, T and NK cells.
	<ol>
		<li>Starting with the whole spleen cell suspension (1 ml of 108&#160;cells/ml obtained in step 2.8), add 100 &#181;l&#160;of biotin-conjugated antibody cocktail provided in the CD8&#945;Pos DC isolation kit.</li>
		<li>Mix well and incubate in ice water bath for 15 min. Tap the tube gently every 3 min&#160;to maximize binding of biotin-labeled antibodies to their target cells.</li>
		<li>Add 12 ml&#160;cell sorting buffer and pellet the cells by centrifugation at 300 x g for 5 min at 4 &#176;C. Aspirate supernatant to remove unbound biotinylated antibodies.</li>
		<li>Add 150 &#181;l&#160;of cell sorting buffer and 100 &#181;l&#160;of anti-biotin beads provided in the CD8&#945;Pos DC isolation kit. Mix well and incubate in ice water bath for 15 min.</li>
		<li>Add 10 ml&#160;cell sorting buffer and pellet the cells by centrifugation at 300 x g for 5 min at 4 &#176;C.</li>
		<li>Resuspend cells in 1 ml&#160;cell sorting buffer and pass over a fresh LS column according to procedure in steps 3.1.6 through 3.1.8.</li>
		<li>Collect the unlabeled flow through 2 (FT2) and discard the column retentate. This unlabeled fraction is enriched for CD8&#945;Pos and CD8&#945;Neg DCs.</li>
		<li>Pellet the cells in FT2 by centrifugation at 300 x g for 5 min at 4 &#176;C.</li>
		<li>Resuspend the cells in 1 ml&#160;cell sorting buffer and add 200 &#181;l&#160;of anti-CD8&#945; conjugated magnetic beads provided in the CD8&#945;Pos DC isolation kit.</li>
		<li>Repeat steps 3.2.2 through 3.2.6. The flow through of the column is enriched for CD8&#945;Neg DCs and depleted for CD8&#945;Pos DCs. This is labeled flow through 3 (FT3).</li>
		<li>To elute the CD8&#945;Pos DCs retained in the column, remove the column from the magnet, add 5 ml&#160;of cell sorting buffer and purge the column matrix using the plunger provided with the LS column.</li>
		<li>Pellet the cells by centrifugation at 300 x g for 5 min at 4 &#176;C. Aspirate supernatant and resuspend in 1 ml&#160;cell sorting buffer. To increase the purity, run the eluted cells again through a fresh LS column by repeating steps 3.1.6 to 3.1.8, except discard the flow through and collect retained cells as in 3.2.11.</li>
		<li>To purify the CD8&#945;Neg DCs from the FT3 suspension, pellet the FT3 by centrifugation at 300 x g for 5 min at 4 &#176;C and resuspend in 300 &#181;l&#160;of cell sorting buffer.</li>
		<li>Add 100 &#181;l&#160;of anti-CD11c magnetic beads. Mix well and incubate in ice water bath for 15 min.</li>
		<li>Add 10 ml&#160;cell sorting buffer and pellet cells by centrifugation at 300 x g for 5 min at 4 &#176;C.</li>
		<li>Repeat steps 3.1.5 through 3.1.8. The CD8&#945;Neg DCs are positively selected with CD11c beads and are bound to the column. The flow through can be discarded.</li>
		<li>Elute the cells retained in the column as described in step 3.2.11 to collect the purified CD8&#945;Neg DCs.</li>
	</ol>
	</li>
</ol>
4. Pulsing APCs with Antigens and Cell Transfer
<ol>
	<li>Count the live cells after purification using trypan blue exclusion 21 to distinguish live and dead cells.</li>
	<li>Incubate the cells with the antigen of choice. Use analogues of glycolipid antigen called alpha-galactosyl&#160;ceramide (&#945;GalCer) at 100 nM concentration13. Typically, plate 106 viable cells/well in complete RPMI medium using ultra-low attachment U-bottom 96 well plates to minimize cell attachment. Incubate the cells in a 5% CO2 atmosphere at 37 &#176;C for 1 - 4 hr.</li>
	<li>Harvest cells by gently pipetting several times. Use wide bore pipette tips to minimize cell damage from shear forces. Wash the wells with PBS and pool these washes to maximize cell harvest.</li>
	<li>Pellet the cells at 300 x g for 5 min at 4 &#176;C and resuspend to desired density in PBS. Typically,&#160;inject 1 million cells in 200 &#181;l&#160;per recipient animal. Keep the cells on ice to maximize viability before administration into host animals.</li>
	<li>Inject cells intravenously into mice either through the lateral tail vain or the retro-orbital venous plexus 19. Use a 27 G needle on a 1 ml&#160;syringe, and inject a maximum volume of 0.1 ml&#160;per mouse.</li>
</ol>

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The authors declare that they have no competing financial interests.

Dendritic cells are accepted to be the major professional antigen presenting cells involved in the priming of T cell responses. Their main function is to survey the tissue microenvironment by taking up and processing antigens for presentation to T cells. In order to study the function of specific DC subsets, these need to be isolated in sufficient numbers using an approach that maintains their normal phenotype and functions. Most protocols rely on the isolation of specific DC subset from na&#239;ve mice. Since these cell...

Dendritic cells (DC) were discovered almost forty years ago as the &#34;large stellate (Greek dendron) cell&#34; found in lymphoid organs1. Many studies have shown that DCs are the only antigen presenting cells that can effectively stimulate na&#239;ve T cells2. A major function of these cells is the uptake and presentation of antigens and their efficient processing and loading of these onto antigen presenting molecules. In mouse spleen, DCs can be separated into plasmacytoid and conventional subsets. The plasmacytoid DCs are characterized by low expression of CD11c and high levels of B220 and Gr-1. They are also positive for the surface marker mPDCA1 and secrete type I interferon in response to toll like receptor 9 (TLR9) ligands. The conventional DCs are high for CD11c and MHC class II expression. They can be split into three distinct subsets based on the surface expression of phenotypic markers such as CD4, CD8&#945;, DEC205, CD11b and dendritic cell inhibitory receptor 2 (DCIR2, recognized by the 33D1 antibody) proteins 3,4. The CD8&#945;Pos DCs are also known as cDC1, are positive for DEC205, but negative for myeloid markers such as CD11b and 33D1. The CD8&#945;Neg DCs, also called cDC2, are positive for 33D1, CD11b and CD4 but lack DEC205. The double negative subset (i.e., negative for both CD4 and CD8&#945;) is relatively rare, and is negative for DEC205 and 33D1. It is the least characterized subset and may be a less differentiated form of CD8&#945;Neg DC.
Phenotypic differences in the various DC subsets also extend to their in vivo functions. The CD8&#945;Neg DCs are highly phagocytic and are thought to present exogenous antigen mainly&#160;via MHC class II to CD4 T cells 3. In contrast, the CD8&#945;Pos DCs are specialized for presentation of soluble protein antigen on MHC class I in a mechanism called cross-presentation. The outcome of cross-presentation depends on the activation status of these DCs5, and can either lead to expansion of cytotoxic T cells (CTLs) or development of regulatory T cells 62,7. Targeting of antigen to CD8&#945;Pos&#160;DCs&#160;using anti-DEC205-antibody-mediated delivery results largely in the deletion of T cells8, whereas presentation of antigens derived from infected apoptotic cells induces a strong CTL response 9.
In addition to recognition of peptide antigens, the mammalian immune system has evolved to recognize lipid and glycolipid antigens. These antigens are presented by CD1 molecules, which are MHC class I-like cell surface proteins that exist in multiple related forms in various mammals. In mice, a single type of highly conserved CD1 molecule called CD1d is responsible for presentation of glycolipid antigens 10. The major population of T cells that recognize CD1d/glycolipid complexes is called invariant NKT cells (iNKT cells). These cells express a semi-invariant T cell receptor (TCR) composed of an invariant TCR&#945; chain that is paired with TCR&#946; chains that have limited diversity 11. Unlike conventional T cells that need to proliferate and differentiate to become activated effector T cells, iNKT cells exist as an effector population and start responding rapidly after glycolipid administration 12. Identification of physiologically relevant lipid antigen presenting cells is an active area of research, and several distinct cell types such as B cells, macrophages and DCs have been suggested to perform this function. However, it was demonstrated that the CD8&#945;Pos subset of DCs is the primary cell mediating uptake and presentation of lipid antigens to mouse iNKT cells 13 and glycolipid mediated cross-priming of CD8 T cells 14.
To compare the efficiency of antigen presentation by different antigen presenting cells, a straightforward approach is to transfer different types of purified APCs pulsed with equivalent amounts of antigen into na&#239;ve hosts. Cell transfer experiments of this type are often performed for immunological studies. However, performing transfer studies with ex vivo antigen treated DCs is challenging, since these cells exist as rare populations in lymphoid organs where they constitute less than 2% of total cells15. It is therefore necessary to enhance the development of these cells in donor animals to increase the efficiency of isolation protocols.
It is known that the common lymphoid and common myeloid progenitors, which are required for generation of pDC, CD8Pos and CD8Neg DC subsets, express fms-related receptor tyrosine kinase 3 (Flt-3). Upon in vivo Flt-3 ligand (Flt-3L) administration, emigration of Flt-3 expressing progenitor cells from bone marrow is increased, resulting in the increased seeding of peripheral lymphoid organs and the expansion of their mature DC progeny16. Expression of Flt-3 is lost during commitment to the B, T or NK cell differentiation pathways. Therefore, only minimal alterations are observed in these cells upon Flt-3L administration. Similar expansion in DC populations is observed in mice bearing tumors generated by implantation of a B16-melanoma cell line secreting murine Flt-3L, which provides a simple and economical method for providing sustained systemic levels of Flt-3L17,18. Using this approach, we have developed a protocol based on the implantation of B16-melanoma cells secreting Flt-3L to stimulate the expansion of all normal DC subsets in mouse spleen, thus greatly increasing the yields of these cells that can be isolated for subsequent experiments. We consistently find that within 10 - 14 days following subcutaneous implantation of the Flt-3 secreting tumor, mice develop splenomegaly with marked enrichment of DCs to constitute 40 - 60% of total spleen cells. From these spleens, different DC subsets can be isolated with high purity using standard commercially available cell purification kits that employ subset-specific phenotypic markers.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.05% Trypsin-EDTA&#160;</td>
			<td>Life Technologies, Gibco</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>CDS019936-250MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase D&#160;</td>
			<td>Roche Diagnostics</td>
			<td>11088858001</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I (dry powder)</td>
			<td>QIAGEN</td>
			<td>79254</td>
			<td></td>
		</tr>
		<tr>
			<td>200 proof ethanol&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>9-6705-004-220</td>
			<td>Used to prepare 70% ethanol</td>
		</tr>
		<tr>
			<td>RBC lysis buffer</td>
			<td>Sigma-Aldrich</td>
			<td>R7757</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI-1640 medium with L-glutamine&#160;</td>
			<td>Life Technologies, Gibco</td>
			<td>11875-119</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM medium with L-glutamine&#160;</td>
			<td>Life Technologies, Gibco</td>
			<td>11995-073</td>
			<td></td>
		</tr>
		<tr>
			<td>200 mM L-glutamine&#160;</td>
			<td>Life Technologies</td>
			<td>25030081</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM non-essential amino acids&#160;</td>
			<td>Life Technologies, Gibco</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM essential amino acids&#160;</td>
			<td>Life Technologies</td>
			<td>11130-051&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-mercaptoethanol&#160;</td>
			<td>Life Technologies, Invitrogen</td>
			<td>21985-023</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate&#160;</td>
			<td>Life Technologies</td>
			<td>11360-070</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES&#160;</td>
			<td>Life Technologies, Invitrogen</td>
			<td>15630</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS Ca2+ and Mg2+ free pH 7.2)</td>
			<td>Life Technologies, Invitrogen</td>
			<td>20012-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s PBS (DPBS with Ca2+ and Mg2+)</td>
			<td>Life Technologies, Gibco</td>
			<td>14040-182</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5 M Ethylenediaminetetraacetate (EDTA) solution&#160;</td>
			<td>Life Technologies</td>
			<td>15575-020</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal calf serum</td>
			<td>Atlanta Biologicals</td>
			<td>S11050&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin&#160;</td>
			<td>Life Technologies, Invitrogen</td>
			<td>15140-163</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue (dry powder)&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>T6146-5G&#160;</td>
			<td>Prepare 0.08% with PBS</td>
		</tr>
		<tr>
			<td>Plasmacytoid Dendritic Cell Isolation Kit II, mouse</td>
			<td>Miltenyi Biotech</td>
			<td>130-092-786</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic beads conjugated with anti-mouse CD11c&#160;</td>
			<td>Miltenyi Biotech</td>
			<td>130-152-001</td>
			<td></td>
		</tr>
		<tr>
			<td>CD8&#945;Pos mouse DC isolation kit&#160;</td>
			<td>Miltenyi Biotech</td>
			<td>130-091-169</td>
			<td></td>
		</tr>
		<tr>
			<td>Fc-gamma receptor blocking antibody (Clone 2.4G2)</td>
			<td>BD Biosciences</td>
			<td>553141</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD11c-FITC&#160;</td>
			<td>BD Biosciences</td>
			<td>553801</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD8&#945;-PerCP&#160;</td>
			<td>BD Biosciences</td>
			<td>553036</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse B220-PE&#160;</td>
			<td>BD Biosciences</td>
			<td>553090</td>
			<td></td>
		</tr>
		<tr>
			<td>1 ml syringes&#160;</td>
			<td>BD</td>
			<td>26048</td>
			<td></td>
		</tr>
		<tr>
			<td>23 G1 needle&#160;</td>
			<td>BD</td>
			<td>305145</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm Petri dishes&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>875712</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical instruments&#160;</td>
			<td>Kent Scientific</td>
			<td>INSMOUSEKIT</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer (70 &#181;m&#160;</td>
			<td>BD</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>Large Petri plates&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>FB0875712</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum filtration system (500 ml(0.22 um&#160;</td>
			<td>Corning</td>
			<td>431097</td>
			<td></td>
		</tr>
		<tr>
			<td>LS columns&#160;</td>
			<td>Miltenyi</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic stand MACS separator&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-302</td>
			<td></td>
		</tr>
		<tr>
			<td>Wide-bore 200 &#956;l pipette tips&#160;</td>
			<td>PerkinElmer</td>
			<td>111623</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning ultra-low attachment 96-well plates&#160;</td>
			<td>Corning</td>
			<td>CLS3474-24EA</td>
			<td></td>
		</tr>
		<tr>
			<td>6-8 week old female C57BL/6 mice&#160;</td>
			<td>Jackson Research Laboratories, Jax</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Murine B16.Flt3L melanoma cell line</td>
			<td></td>
			<td></td>
			<td>described by Mach et al. ,2000</td>
		</tr>
		<tr>
			<td>Serum free DMEM and RPMI media</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>500 ml DMEM with L-glutamine, or 500 ml RPMI-1640 with L-glutamine 
			 
			5 ml MEM nonessential amino acids (100x, 10 mM)&#160;&#160;&#160;&#160; 
			 
			5 ml HEPES Buffer (1 M)&#160;&#160;&#160;&#160; 
			 
			5 ml L-glutamine (200 mM)&#160; 
			 
			0.5 ml 2-mercaptoethanol (5.5 x 10-2 M)</td>
			<td></td>
			<td></td>
			<td>Mix all the ingredients in a biosafety cabinet with either DMEM or RPMI media depending on your need.&#160; Filter sterilize the media by passing through a 0.22 &#181;m vacuum filtration system.</td>
		</tr>
		<tr>
			<td>Complete RPMI and DMEM media</td>
			<td></td>
			<td></td>
			<td>Add 50 ml of heat inactivated fetal calf serum to serum free RPMI or DMEM media to obtain complete media.</td>
		</tr>
		<tr>
			<td>MACS buffer:&#160;</td>
			<td></td>
			<td></td>
			<td>Add 2 ml of 0.5 M EDTA and 10 ml of heat inactivated fetal calf serum to 500 ml of Phosphate-buffered saline (PBS, Ca2+ and Mg2+ Free, pH 7.2) and 2 ug/ml 2.4G2 (Fc-Block antibody).&#160;</td>
		</tr>
		<tr>
			<td>FACS staining buffer</td>
			<td></td>
			<td></td>
			<td>Dissolve sodium azide to 0.05% (0.25 g per 500 ml) in MACS buffer to obtain FACS staining buffer</td>
		</tr>
		<tr>
			<td>10x Collagenase D and DNase 1 stock solution 
			Dissolve 1 g of collagenase D and 0.2 ml of DNase 1 stock (1 mg/ml, 100x) in 20 ml of PBS containing Ca++ and Mg++ to obtain a solution of approximately 1,000-</td>
			<td></td>
			<td></td>
			<td>2,000 Units of collagenase activity per ml 
			 
			This 10x stock solution can be prepared ahead of time and stored at -20 &#176;C for several weeks. Dilute 1 ml of this with 9 ml of serum free RPMI immediately before use</td>
		</tr>
	</tbody>
</table>

an efficient and high yield method for isolation of mouse dendritic cell subsets

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen presenting molecules to initiate T-cell-mediated immunity. Dendritic cells can be separated into several phenotypically and functionally heterogeneous subsets. Three important subsets of splenic dendritic cells are plasmacytoid, CD8&#945;Pos and CD8&#945;Neg cells. The plasmacytoid DCs are natural producers of type I interferon and are important for anti-viral T cell immunity. The CD8&#945;Neg DC subset is specialized for MHC class II antigen presentation and is centrally involved in priming CD4 T cells. The CD8&#945;Pos DCs are primarily responsible for cross-presentation of exogenous antigens and CD8 T cell priming. The CD8&#945;Pos DCs have been demonstrated to be most efficient at the presentation of glycolipid antigens by CD1d molecules to a specialized T cell population known as invariant natural killer T (iNKT) cells. Administration of Flt-3 ligand increases the frequency of migration of dendritic cell progenitors from bone marrow, ultimately resulting in expansion of dendritic cells in peripheral lymphoid organs in murine models. We have adapted this model to purify large numbers of functional dendritic cells for use in cell transfer experiments to compare in vivo proficiency of different DC subsets.

The outcome of this procedure relies on the expansion of DC subsets by murine Flt-3L expressed by the implanted melanoma cells. The B16.Flt3L tumor was derived from a C57BL/6 mouse, and should be implanted into animals with that strain background in order to avoid failure of the tumor to become established due to rejection. In some cases, it may be desirable to use genetically modified mice to derive DCs with known defects in signaling pathways or receptors of interest. It is important to...

Watch this Scientific Journal Video about An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets at JoVE.com

An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets

Distinct dendritic cell subsets exist as rare populations in lymphoid organs, and therefore are challenging to isolate in sufficient numbers and purity for immunological experiments. Here we describe a high efficiency, high yield method for isolation of all of the currently known major subsets of mouse splenic dendritic cells.

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen ...

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Research

JoVE Journal

Immunology and Infection

15.1K Views.  Albert Einstein College of Medicine. Distinct dendritic cell subsets exist as rare populations in lymphoid organs, and therefore are challenging to isolate in sufficient numbers and purity for immunological experiments. Here we describe a high efficiency, high yield method for isolation of all of the currently known major subsets of mouse splenic dendritic cells.

Video: An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets

Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection of pathogenic antigen by na&iuml;ve DCs is through pattern recognition receptors (PRRs) which are able to recognize specific conserved structures referred to as pathogen-associated molecular patterns (PAMPS). Detection of PAMPs by DCs triggers an intracellular signaling cascade resulting in their activation and transformation to mature DCs. This process is typically characterized by production of type 1 interferon along with other proinflammatory cytokines, upregulation of cell surface markers such as MHCII and CD86 and migration of the mature DC to draining lymph nodes, where interaction with T cells initiates the adaptive immune response2,3. Thus, DCs link the innate and adaptive immune systems. 
The ability to dissect the molecular networks underlying DC response to various pathogens is crucial to a better understanding of the regulation of these signaling pathways and their induced genes. It should also help facilitate the development of DC-based vaccines against infectious diseases and tumors. However, this line of research has been severely impeded by the difficulty of transfecting primary DCs4.
Virus transduction methods, such as the lentiviral system, are typically used, but carry many limitations such as complexity and bio-hazardous risk (with the associated costs)5,6,7,8. Additionally, the delivery of viral gene products increases the immunogenicity of those transduced DCs9,10,11,12. Electroporation has been used with mixed results13,14,15, but we are the first to report the use of a high-throughput transfection protocol and conclusively demonstrate its utility.
In this report we summarize an optimized commercial protocol for high-throughput transfection of human primary DCs, with limited cell toxicity and an absence of DC maturation16. Transfection efficiency (of GFP plasmid) and cell viability were more than 50% and 70% respectively. FACS analysis established the absence of increase in expression of the maturation markers CD86 and MHCII in transfected cells, while qRT-PCR demonstrated no upregulation of IFN&beta;. Using this electroporation protocol, we provide evidence for successful transfection of DCs with siRNA and effective knock down of targeted gene RIG-I, a key viral recognition receptor16,17, at both the mRNA and protein levels.

We present our optimized high-throughput nucleofection protocol as an efficient way of transfecting primary human monocyte-derived dendritic cells with either plasmid DNA or siRNA without causing cell maturation. We further provide evidence for successful siRNA silencing of targeted gene RIG-I at both the mRNA and protein levels.

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection ...

Isolation of Mouse Lung Dendritic Cells

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the respiratory tract. At least three main subsets of lung dendritic cells have been described in mice: conventional DC (cDC) 4, plasmacytoid DC (pDC) 5 and the IFN-producing killer DC (IKDC) 6,7. The cDC subset is the most prominent DC subset in the lung 8. 
The common marker known to identify DC subsets is CD11c, a type I transmembrane integrin (&beta;2) that is also expressed on monocytes, macrophages, neutrophils and some B cells 9. In some tissues, using CD11c as a marker to identify mouse DC is valid, as in spleen, where most CD11c+ cells represent the cDC subset which expresses high levels of the major histocompatibility complex class II (MHC-II). However, the lung is a more heterogeneous tissue where beside DC subsets, there is a high percentage of a distinct cell population that expresses high levels of CD11c bout low levels of MHC-II. Based on its characterization and mostly on its expression of F4&#47;80, an splenic macrophage marker, the CD11chiMHC-IIlo lung cell population has been identified as pulmonary macrophages 10 and more recently, as a potential DC precursor 11. 
In contrast to mouse pDC, the study of the specific role of cDC in the pulmonary immune response has been limited due to the lack of a specific marker that could help in the isolation of these cells. Therefore, in this work, we describe a procedure to isolate highly purified mouse lung cDC. The isolation of pulmonary DC subsets represents a very useful tool to gain insights into the function of these cells in response to respiratory pathogens as well as environmental factors that can trigger the host immune response in the lung.

A highly purified preparation of mouse lung dendritic cells is described. Specific emphasis is given to the isolation of conventional dendritic cell subset.

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the ...

Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and food antigens while concomitantly remaining poised to mount inflammatory responses toward invasive pathogens1,2. Antigen presenting cells, particularly DCs and macrophages, play critical roles in maintaining intestinal immune homeostasis via their ability to sense and appropriately respond to the microbiota3-14. Efficient isolation of intestinal DCs and macrophages is a critical step in characterizing the phenotype and function of these cells. While many effective methods of isolating intestinal immune cells, including DCs and macrophages, have been described6,10,15-24, many rely upon long digestions times that may negatively influence cell surface antigen expression, cell viability, and/or cell yield. Here, we detail a methodology for the rapid isolation of large numbers of viable, intestinal DCs and macrophages. Phenotypic characterization of intestinal DCs and macrophages is carried out by directly staining isolated intestinal cells with specific fluorescence-labeled monoclonal antibodies for multi-color flow cytometric analysis. Furthermore, highly pure DC and macrophage populations are isolated for functional studies utilizing CD11c and CD11b magnetic-activated cell sorting beads followed by cell sorting.

Here, we detail a methodology for the rapid isolation of mouse intestinal dendritic cells (DCs) and macrophages. Phenotypic characterization of intestinal DCs and macrophages is performed using multi-color flow cytometric analysis while magnetic bead enrichment followed by cell sorting is used to yield highly pure populations for functional studies.

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and ...

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for isolating precursor B-cells at four different stages of differentiation. Because genes are expressed and epigenetic modifications occur in a tissue specific manner, it is vital to discriminate between tissues and cell types in order to be able to identify alterations in the genome and the epigenome that may lead to the development of disease. This method can be adapted to any type of cell present in umbilical cord blood at any stage of differentiation.	
This method comprises 4 main steps. First, mononuclear cells are separated by density centrifugation. Second, B-cells are enriched using biotin conjugated antibodies that recognize and remove non B-cells from the mononuclear cells. Third the B-cells are fluorescently labeled with cell surface protein antibodies specific to individual stages of B-cell development. Finally, the fluorescently labeled cells are sorted and individual populations are recovered. The recovered cells are of sufficient quantity and quality to be utilized in downstream nucleic acid assays.

Here we describe a protocol for isolating subsets of precursor B-cells from umbilical cord blood. A sufficient quantity and quality of nucleic acids may be extracted from the cells and used in subsequent assays utilizing DNA or RNA.

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of free merozoites from the blood by phagocytic cells. However assessing the functional efficacy of merozoite specific opsonizing antibodies is challenging due to the short half-life of merozoites and the variability of primary phagocytic cells. Described in detail herein is a method for generating viable merozoites using the E64 protease inhibitor, and an assay of merozoite opsonin-dependent phagocytosis using the pro-monocytic cell line THP-1. E64 prevents schizont rupture while allowing the development of merozoites which are released by filtration of treated schizonts. &#160;Ethidium bromide labelled merozoites are opsonized with human plasma samples and added to THP-1 cells. Phagocytosis is assessed by a standardized high throughput protocol. Viable merozoites are a valuable resource for assessing numerous aspects of P. falciparum biology, including assessment of immune function. Antibody levels measured by this assay are associated with clinical immunity to malaria in naturally exposed individuals. The assay may also be of use for assessing vaccine induced antibodies. &#160;

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

Measuring antibody function is key to understanding immunity to Plasmodium falciparum malaria. This method describes the purification of viable merozoites, and measurement of opsonization-dependent phagocytosis by flow cytometry.

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of ...

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition of cognate antigen presented on MHC class II. The cytokine signals present in the environment at the time of TCR activation are a major factor in determining the effector fate of a na&#239;ve CD4+ T cell. Although the cytokine environment during na&#239;ve T cell activation may be complex and involve both redundant and opposing signals in vivo, the addition of various cytokine combinations during naive CD4+ T cell activation in vitro can readily promote the establishment of effector T helper lineages with hallmark cytokine and transcription factor expression. Such differentiation experiments are commonly used as a first step for the evaluation of targets believed to promote or inhibit the development of certain CD4+ T helper subsets. The addition of mediators, such as signaling agonists, antagonists, or other cytokines, during the differentiation process can also be used to study the influence of a particular target on T cell differentiation. Here, we describe a basic protocol for the isolation of na&#239;ve T cells from mouse and the subsequent steps necessary for polarizing na&#239;ve cells to various T helper effector lineages in vitro.

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Na&#239;ve CD4+ T cells polarize to various subsets depending on the environment at the time of activation. The differentiation of na&#239;ve CD4+ T cells to various effector subsets can be achieved in vitro through the addition of T cell receptor stimuli and specific cytokine signals.

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. Regulatory systems that utilize intracellular cyclic di-GMP (c-di-GMP) as a second messenger are one such class of target. c-di-GMP is a signaling molecule found in almost all bacteria that acts to regulate an extensive range of processes including antibiotic resistance, biofilm formation and virulence. The understanding of how c-di-GMP signaling controls aspects of antibiotic resistant biofilm development has suggested approaches whereby alteration of the cellular concentrations of the nucleotide or disruption of these signaling pathways may lead to reduced biofilm formation or increased susceptibility of the biofilms to antibiotics. We describe a simple high-throughput bioreporter protocol, based on green fluorescent protein (GFP), whose expression is under the control of the c-di-GMP responsive promoter cdrA, to rapidly screen for small molecules with the potential to modulate c-di-GMP cellular levels in Pseudomonas aeruginosa (P. aeruginosa). This simple protocol can screen upwards of 3,500 compounds within 48 hours and has the ability to be adapted to multiple microorganisms.

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

This article describes the high-throughput assay that has been successfully established to screen large libraries of small molecules for their potential ability to manipulate cellular levels of cyclic di-GMP in Pseudomonas aeruginosa, providing a new powerful tool for antibacterial drug discovery and compound testing.

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method enables researchers to better understand the characteristics of a single cell population without the influence of other cells. Compared to other methods of cell enrichment, such as magnetic-activated cell sorting (MCS), FACS is more flexible and accurate for cell separation due to the ability of phenotype detection by flow cytometry. In addition, FACS is usually capable of separating multiple cell populations simultaneously, which improves the efficiency and diversity of experiments. Although FACS has some limitations, it has been broadly used to purify cells for functional studies in both in vitro and in vivo settings. Here we report a protocol using fluorescence-activated cell sorting to isolate a very rare population of immune cells, plasmacytoid dendritic cells (pDC), with high purity from the bone marrow of lupus-prone mice for in vitro functional studies of pDC.

We report a protocol using fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells (pDC) with high purity from the bone marrow of lupus-prone mice for functional studies of pDC.

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets

Summary

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Chapters in this video

Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation

Isolation of Mouse Lung Dendritic Cells

Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

Mouse Naïve CD4⁺ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow