We thank Flow Cytometry Laboratory at Virginia-Maryland College of Veterinary Medicine for the use of flow cytometry core facility. This work was supported by XML's startup funds. XL is a Stamps Fellow in the Biomedical and Veterinary Sciences graduate program.

NOTE: MRL/Mp-Faslpr (MRL/lpr) lupus-prone mice were bred and maintained in a specific pathogen-free facility following the requirements of Institutional Animal Care and Use Committee (IACUC) at Virginia Tech (Animal Welfare Assurance Number: A3208-01). This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All animal experiments were conducted under IACUC protocol #12-062.
1. Cell Culture Medium and Sorting Buffer
<ol>
	<li>Prepare complete cell culture medium (C10) using RPMI 1640 supplemented with 10% fetal bovine serum, 1 mM sodium pyruvate, 1% 100 MEM non-essential amino acids, 10 mM HEPES, 55 &#181;M 2-mercaptoethanol, 2 mM L-glutamine and 100 U/ml penicillin-streptomycin.</li>
	<li>Prepare sorting buffer (HBSS-full) using 1x Hank&#39;s Balanced Salt Solution (HBSS) supplemented with 10 mM HEPES, 2.5 mg/ml bovine serum albumin, 0.05 mM MgCl2 and 0.2 U/ml DNase I.</li>
</ol>
2. Mouse Dissection
<ol>
	<li>Prepare two 6-cm diameter dishes containing 4 ml C10. Place the dishes on ice.</li>
	<li>Euthanize the mouse by CO2 inhalation.</li>
	<li>Harvest the spleen and keep it in a dish with C10 on ice.
	<ol>
		<li>Pin the mouse down with the belly facing up on the dissection plate. Sterilize the body by spraying 70% ethanol.</li>
		<li>Cut the skin and separate the skin from the muscle wall underneath along the ventral midline from the pubic symphysis to the neck.</li>
		<li>Cut the skin along the hind limb from the first incision to the ankle.</li>
		<li>Pin the skin back on the sides and cut the muscle wall along the sides from the first incision place to diaphragm.</li>
		<li>Pin the muscle wall back on the right side of the head.</li>
		<li>Locate the spleen on the right side of the mouse underneath the small intestine and beside the stomach. Pick up one end of the spleen and separate it from the stomach.</li>
	</ol>
	</li>
	<li>Cut both hind limbs out, including femur and tibia, and remove all muscles as much as possible.
	<ol>
		<li>Cut the muscles along the hind limb from the pelvic/hip joint to the ankle with a sharp scissor, trying to avoid blood vessels.</li>
		<li>Separate the hind limb out by cutting at the pelvic/hip joint and ankle/foot joint without the exposure of bone marrow contents.</li>
		<li>Clean remaining muscles on the bones by scratching with a razor blade repeatedly and cutting the muscles at the joint points.</li>
	</ol>
	</li>
	<li>Keep the bones in a 6 cm dish containing 4 ml C10 on ice. Proceed to dissecting the next mouse.</li>
</ol>
3. Splenocyte Isolation
<ol>
	<li>Homogenize the spleen gently with a syringe plunger against a 70-&#181;m cell strainer on top of the dish containing 4 ml C10 until no red pieces are visible.</li>
	<li>Add additional 6 ml C10 into the dish through the strainer and then transfer the total 10 ml cell suspension to a 15 ml conical tube.</li>
	<li>Centrifuge the conical tube at 800 x g for 5 min, 4 &#176;C.</li>
	<li>After centrifugation, aspirate the supernatant and resuspend the cell pellet in 2 ml 1 x red blood cell (RBC) lysis buffer. Incubate at RT for 5 min.</li>
	<li>After incubation, centrifuge the conical tube at 800 x g for 5 min, 4 &#176;C.</li>
	<li>After centrifugation, aspirate the supernatant and resuspend the cell pellet in 1 ml C10. Discard any big clumps (dead cells) and keep the tube on ice for later. 
	NOTE: If there are many small clumps, use a 70-&#181;m cell strainer to filter the cell suspension once.</li>
	<li>Count the cell number with trypan blue using an automated cell counter.</li>
</ol>
4. Bone Marrow Cell Isolation
<ol>
	<li>Transfer the bones with 4 ml C10 into a mortar and add 6 ml C10 to make a volume of 10 ml C10 in total.</li>
	<li>Crack the bones gently in the mortar by using a pestle.</li>
	<li>Stir gently with the pestle to release the bone marrow into C10.</li>
	<li>Transfer the 10 ml C10 containing bone marrow into a 50 ml conical tube on ice through a 70-&#181;m cell strainer. Add 10 ml fresh C10 into the mortar still containing the bones. 
	NOTE: Pipette the solution containing bone marrow up and down several times before passing through the strainer if red clumps are visible.</li>
	<li>Repeat steps 4.2 to 4.4 twice. After the last wash, the bones should appear white.</li>
	<li>Centrifuge the 50 ml conical tube at 800 x g for 5 min, 4 &#176;C. Meanwhile, prepare 5 ml ready-to-use density gradient medium in a 15 ml conical centrifuge tube at RT.</li>
	<li>After centrifugation, aspirate the supernatant and resuspend the cell pellet in 10 ml C10.</li>
	<li>Slowly layer the 10 ml cell suspension on top of the 5 ml density gradient medium, maintaining clear interface between the two phases. Then centrifuge the 15 ml conical tube at 1,363 g for 30 min at RT (20 &#176;C) with acceleration set as 9 and deceleration set as 0 (or Brake OFF).</li>
	<li>After centrifugation, remove the top 8 ml solution carefully and collect the 2 ml buffy coat at the interface (a layer of mononuclear cells) into a new 15 ml conical tube.</li>
	<li>Add 12 ml ice cold C10 to the 15 ml conical tube containing bone marrow mononuclear cells, cap and invert several times to mix well, then centrifuge the tube at 800 x g for 10 min, 4 &#176;C.</li>
</ol>
5. Cell Surface Staining for FACS
<ol>
	<li>Sample staining
	<ol>
		<li>Prepare anti-mouse CD16/32 antibody solution (1 to 100 dilution in HBSS-full, 5 &#181;g/ml final concentration).</li>
		<li>After centrifugation in step 4.10, aspirate the supernatant and resuspend the cell pellet in 100 &#181;l anti-mouse CD16/32 antibody solution to block Fc receptor on the mononuclear cells. Incubate on ice for 10 min.</li>
		<li>Prepare fluorescence-conjugated antibody mixture (anti-mouse CD11c-PE 1:40 dilution, anti-mouse CD11b-APC-CY7 1:80 dilution, anti-mouse PDCA-1-FITC 1:40 dilution and anti-mouse B220-V500 1:40 dilution in HBSS-full as recommended by the manufacturer at 100 &#181;l final volume per sample). 
		NOTE: It is important to titrate antibodies before use.</li>
		<li>After 10 min incubation in step 5.1.2, add 4 ml ice cold HBSS-full and centrifuge at 800 x g for 5 min, 4 &#176;C to remove unbound anti-mouse CD16/32.</li>
		<li>After centrifugation, aspirate the supernatant and resuspend the cell pellet in 100 &#181;l fluorescence-conjugated antibody mixture. Incubate on ice for 15 min in the dark.</li>
		<li>After 15 min incubation, add 4 ml ice cold HBSS-full and centrifuge at 800 x g for 5 min, 4 &#176;C to remove unbound fluorescence-conjugated antibodies.</li>
		<li>Prepare FACS loading solution (500 ng/ml DAPI in HBSS-full).</li>
		<li>After centrifugation in step 5.1.6, aspirate the supernatant and resuspend the cell pellet in 500 &#181;l FACS loading solution. Transfer the cell suspension into a 12 x 75 mm round bottom tube (FACS tube) and keep the tube on ice in the dark until sorting within 1 hr.</li>
	</ol>
	</li>
	<li>Staining for compensation
	<ol>
		<li>Label 6 FACS tubes as PE, APC-CY7, FITC, V500, DAPI or unstained. Aliquot splenocytes at 1 x 106 cells/tube into the FACS tubes, and wash with 4 ml HBSS-full at 800 x g for 5 min, 4 &#176;C. 
		NOTE: Use DNA-binding fluorescence dye, DAPI, to distinguish live cells from dead cells. DAPI can pass through the plasma membrane of dead cells (but not of live cells) and bind chromosome DNA.</li>
		<li>Aspirate the supernatant and resuspend the cell pellet in 100 &#181;l HBSS-full.</li>
		<li>In each corresponding FACS tube, add one of the following antibodies: anti-mouse CD19-PE 1:40 dilution, anti-mouse CD11b-APC-CY7 1:80 dilution, anti-mouse PDCA-1-FITC 1:40 dilution or anti-mouse B220-V500 1:40 dilution as recommended by the manufacturer. Leave the 5th (DAPI) and 6th (unstained) FACS tubes as they are. Mix well by shaking and incubate all the FACS tubes on ice in the dark for 15 min.</li>
		<li>After incubation, add 4 ml ice cold HBSS-full into each tube and centrifuge the FACS tubes at 800 x g for 5 min, 4 &#176;C.</li>
		<li>After centrifugation, aspirate the supernatant and resuspend the cell pellet in 500 &#181;l ice cold HBSS-full except for the pellet in the tube labeled DAPI, which is resuspended in 500 &#181;l FACS loading solution. Keeping all 6 tubes on ice in the dark until sorting.</li>
	</ol>
	</li>
</ol>
6. Sorting on the Cell Sorter
NOTE: Operation of sorting procedure on the cytometer and software is standardized with a detailed instruction provided by the company. Briefly, we use 100 micron nozzle at 20 psi, set target cell concentration between 5 - 10 million per ml, and adjust efficiency to 70% or higher (i.e., conflicts kept under 30%).
<ol>
	<li>Prepare collection FACS tubes with 500 &#181;l FBS/tube containing 100 U/ml penicillin-streptomycin.</li>
	<li>Adjust compensation parameters of the cell sorter using single-stain compensation tubes from step 5.2.
	<ol>
		<li>Record 10,000 cells in unstained tube to set negative gate for each fluorescent intensity.</li>
		<li>Record 10,000 cells in other single-stain compensation tubes to set positive gate for each fluorescent intensity. 
		NOTE: The software automatically calculates the compensation parameters.</li>
	</ol>
	</li>
	<li>Use one sample from step 5.1 to set the gates of sorted cell populations by recording 3,000 cells. Using fluorescent intensity as parameters of X and Y-axis graph, gate target cell population manually as a group of dots apparently separated from other dots. 
	NOTE: This gating set is flexible and arbitrary based on the requirements of researchers. If researchers expect higher purity based on one or more markers, move the gate higher based on the corresponding fluorescent intensity.</li>
	<li>Load a collection tube and start to sort the target cell population.</li>
	<li>Keep the collection tube on ice after sorting until all sample tubes are done.</li>
	<li>Add ice cold C10 to the collection tube up to 4 ml, cap and invert to mix.</li>
	<li>Centrifuge the collection tube at 800 x g for 5 min, 4 &#176;C, and then aspirate the supernatant, leaving around 200 &#181;l with cell pellet untouched.</li>
	<li>Add 1 ml ice cold C10 to resuspend the cell pellet and transfer all cell suspension into a 1.5 ml centrifuge tube.</li>
	<li>Centrifuge the 1.5 ml tube at 800 x g for 5 min, 4 &#176;C and aspirate the supernatant, leaving 100 &#181;l with the cell pellet untouched.</li>
	<li>Store the sorted cell populations on ice until use.</li>
</ol>

<ol>
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D., Farsund, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+application+of+flow+cytometry:+a+review.">Clinical application of flow cytometry: a review.</a> Cytometry. 2 (1), 1-13 (1981).</li><li>Alvarez-Barrientos, A., Arroyo, J., Canton, R., Nombela, C., Sanchez-Perez, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+flow+cytometry+to+clinical+microbiology.">Applications of flow cytometry to clinical microbiology.</a> Clin Microbiol Rev. 13 (2), 167-195 (2000).</li><li>Mattanovich, D., Borth, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+cell+sorting+in+biotechnology.">Applications of cell sorting in biotechnology.</a> Microb Cell Fact. 5 (12), (2006).</li><li>Aldahlawi, A. M., Elshal, M. F., Damiaiti, L. A., Damanhori, L. H., Bahlas, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+CD95+and+CCR7+expression+on+circulating+CD4(+)+lymphocytes+revealed+disparate+immunoregulatory+potentials+in+systemic+lupus+erythematosus.">Analysis of CD95 and CCR7 expression on circulating CD4(+) lymphocytes revealed disparate immunoregulatory potentials in systemic lupus erythematosus.</a> Saudi J Biol Sci. 23 (1), 101-107 (2016).</li><li>Cullender, T. C., 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=Innate+and+adaptive+immunity+interact+to+quench+microbiome+flagellar+motility+in+the+gut.">Innate and adaptive immunity interact to quench microbiome flagellar motility in the gut.</a> Cell Host Microbe. 14 (5), 571-581 (2013).</li><li>Fernandez, A. G., 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=High-throughput+fluorescence-based+isolation+of+live+C.+elegans+larvae.">High-throughput fluorescence-based isolation of live C. elegans larvae.</a> Nat Protoc. 7 (8), 1502-1510 (2012).</li><li>Cai, 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=DC-SIGN+expression+on+podocytes+and+its+role+in+inflammatory+immune+response+of+lupus+nephritis.">DC-SIGN expression on podocytes and its role in inflammatory immune response of lupus nephritis.</a> Clin Exp Immunol. 183 (3), 317-325 (2016).</li><li>Blasius, A., 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=A+cell-surface+molecule+selectively+expressed+on+murine+natural+interferon-producing+cells+that+blocks+secretion+of+interferon-alpha.">A cell-surface molecule selectively expressed on murine natural interferon-producing cells that blocks secretion of interferon-alpha.</a> Blood. 103 (11), 4201-4206 (2004).</li><li>Welzel, G., Seitz, D., Schuster, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic-activated+cell+sorting+(MCS)+can+be+used+as+a+large-scale+method+for+establishing+zebrafish+neuronal+cell+cultures.">Magnetic-activated cell sorting (MCS) can be used as a large-scale method for establishing zebrafish neuronal cell cultures.</a> Sci Rep. 5, 7959 (2015).</li><li>Valli, H., 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=Fluorescence-+and+magnetic-activated+cell+sorting+strategies+to+isolate+and+enrich+human+spermatogonial+stem+cells.">Fluorescence- and magnetic-activated cell sorting strategies to isolate and enrich human spermatogonial stem cells.</a> Fertil Steril. 102 (2), 566-580 (2014).</li><li>Yamashiro, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+and+functional+change+of+cytokine-activated+neutrophils:+inflammatory+neutrophils+are+heterogeneous+and+enhance+adaptive+immune+responses.">Phenotypic and functional change of cytokine-activated neutrophils: inflammatory neutrophils are heterogeneous and enhance adaptive immune responses.</a> J Leukoc Biol. 69 (5), 698-704 (2001).</li><li>Apostolidis, S. A., Lieberman, L. A., Kis-Toth, K., Crispin, J. C., Tsokos, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dysregulation+of+cytokine+networks+in+systemic+lupus+erythematosus.">The dysregulation of cytokine networks in systemic lupus erythematosus.</a> J Interferon Cytokine Res. 31 (10), 769-779 (2011).</li><li>Asselin-Paturel, C., Brizard, G., Pin, J. 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The authors declare that there is no conflict of interest regarding the publication of this paper.

The protocol described in this manuscript is for high purity enrichment of live pDC that retain the ability to produce IFN&#945;. The applications of this protocol include, but are not limited to, purification of pDC and/or any other mononuclear cells from the bone marrow of MRL/lpr and any other mouse strains for studies of cellular and molecular functions. Several critical steps in this protocol are to ensure high viability and purity of the sorted pDC. The first key step is the release of bone marrow from bones. To minimize damaging bone marrow cells, it is important to gently crack the bones and stir the pestle instead of smashing the bones. The second key step is the enrichment of mononuclear cells with density gradient medium. It is critical to establish a clear interface between the lower phase (density gradient medium) and the upper phase (cell suspension) while loading the cell suspension. The centrifugation condition is also critical for the formation of the buffy coat layer that contains mainly mononuclear cells. After centrifugation, bone debris, red blood cells and high-density neutrophils are excluded from the buffy coat layer and stay at the bottom of the tube. The third key step is incubating the cells with fluorescence-conjugated antibodies at a low temperature (on ice or 4 &#176;C). After the staining, it is also important to wash the cells extensively to ensure the specific antibody binding with minimal false positives or background signals easily detectable by the cell sorter. Finally, the single-stain compensation tubes are important and necessary for the adjustment of compensation parameters to ensure only the correct signals are measured and analyzed on the cell sorter. Besides using isolated splenocytes for the compensation, commercially available compensation beads can be an alternative if cells are limited. In this particular study, as we needed to enrich as many pDC as possible for further functional studies, to avoid bone marrow cell loss, we used splenocytes instead of bone marrow cells for the compensation. Also, it needs to be noted that surface marker like CD11c are expressed on rare populations, so they are not the best choice for compensation. Instead, an alternative marker expressed at a relatively high level on a major population can be used for compensation purpose as long as the antibody is conjugated with the same fluorescence dye. For example, in this study, CD19-PE is used to replace CD11c-PE in the compensation staining of splenocytes. Besides single color controls, fluorescence minus one (FMO) controls are important for beginners, especially to sort a population with markers not apparently separated from other populations. However, in this study, single color controls are adequate as pDC are a distinct population clearly defined and separated from other cells. In addition, we could set the gate slightly smaller than the actual population to improve purity.
In our studies, we only sorted a single population by FACS. In fact, this sorting method can be used broadly not only to enrich several different cell populations simultaneously, but also to isolate any interesting particle targets within a certain size range decided by the cell sorter, as long as they can be labeled with fluorescence. MCS is another cell enrichment method similar to FACS by principle that both are based on antibody detection of cell surface markers. Even though MCS is also widely used, it may not be efficient in various situations. For example, the phenotype of a cell population may change with the microenvironment in vivo or culture condition in vitro. MCS is prone to errors when the cell phenotype changes, as it usually detects only one surface molecule. On the contrary, FACS is based on the combination of several adjustable markers that reflect a more specific and accurate phenotype of a cell population in dynamic conditions. In addition, the gating strategy of FACS gives it a more sophisticated sorting ability, such as sorting Ly6C+ monocytes into Ly6Chigh and Ly6Cmedium subsets, which cannot be done with MCS. 
However, FACS has its limitations as well. As it separates cell populations according to the analysis of fluorescent signals, if the cells or particles of interest have high autofluorescence, it will be difficult to sort the targets. In addition, a designated professional staff is usually required to operate the cell sorter properly. Moreover, the binding of fluorescence-conjugated antibodies to their specific ligands on cells may change the function of sorted cells, affecting the outcomes of subsequent experiments. For example, Siglec-H as a pDC specific surface molecule can be used to sort pDC, but the binding of Siglec-H by its specific antibody has been demonstrated to suppress the ability of pDC to produce IFN&#945;. Therefore, selection of markers for cell sorting needs to be cautious and based on the purpose of the study. 
Furthermore, the high-throughput cell sorter is limited to sorting, with high efficiency, only target cells of certain percentage within the total cell population. In this study, we first used density gradient centrifugation to increase the percentage of pDC through removing red blood cells, high dense granulocytes and debris, which was a better choice than simply adding the red blood cell lysis buffer. If the population is so rare that is less than 1%, the sorting efficiency will drop with significant cell loss. Hence, extremely rare population can be enriched first by other methods, such as negative selection with MCS to remove some of the other cells, and then sorted by FACS to achieve high purity with minimal cell loss. In summary, FACS is an efficient and accurate method to enrich functional pDC from the mouse bone marrow with high purity.

Efficient separation of a cell population of choice from other cells enables studies of the population that may not be possible otherwise. Fluorescence-activated cell sorting (FACS) is a method to enrich an interesting cell population with high purity. 1,2 Different cell types usually express unique molecules, or a unique combination of several molecules, on the plasma membrane that can distinguish one cell population from another. Upon binding of these cell surface molecules by specific fluorescence-conjugated antibodies, a detecting machine called flow cytometer/sorter is able to excite and detect the light signals of different fluorescent dyes that represent different molecule markers on the cells at the single cell level. The combined information consisting of either the presence of a light signal (representing positive expression of the corresponding surface molecule) or the absence of a light signal (representing negative expression of a molecule) defines the phenotype of the cell. After passing through the detector, cells with the same phenotype of interest are diverted towards a designated collecting tube based on electrical charge.
FACS is broadly applied in various studies as long as the population to be enriched is labeled with fluorescence.3-7 It has been used to separate immunoglobulin (Ig)A-coated bacteria from non-IgA coated bacteria in the gut microbiota 8 and sort genetically engineered cell populations expressing fluorescent proteins. 9 Importantly, it has the capacity to separate more than one population simultaneously, which not only saves time and reagents but also allows for more sophisticated study designs. 10 However, FACS also has its limitations. If a population of interest is very rare (less than 1%), the sorting efficiency may be reduced, causing significant cell loss. In addition, some antibody binding may activate intracellular signal transduction that induces functional changes of the sorted cell population. 11 Therefore, the phenotype used for sorting should be selected carefully. 
Other methods exist besides FACS that are also based on cell surface markers for the enrichment of specific cell populations, such as magnetic-activated cell sorting (MCS). 12 Similar to FACS, magnetic beads-conjugated antibodies can target specific cell surface molecules. Upon antibody-antigen interaction, magnetic beads-coated cells can be separated from non-coated cells after passing through a magnetic field. However, only a limited number of molecules can be targeted in MCS, as magnetic beads are, unlike various fluorescent colors in FACS, undistinguishable. It is thus difficult for MCS to define a cell phenotype with a complicated combination of surface markers. 13,14 In addition, MCS is also able to cause unintended activation of target cells.
In our studies of a mouse model of systemic lupus erythematosus (SLE), 15 we intended to purify plasmacytoid dendritic cells (pDC) to investigate their functional changes with disease progression. We first used MCS to enrich pDC from the bone marrow by targeting PDCA-1, a molecule highly and uniquely expressed on murine pDC at steady state. 16 However, the cell purity was unexpectedly low, likely due to the upregulation of PDCA-1 on other cell populations in an inflammatory environment such as SLE.16 Ultimately, we have used FACS with a combination of four surface markers (CD11c, CD11b, B220 and PDCA-1) to separate high-purity pDC as CD11c+CD11b-B220+PDCA-1+ population. Murine pDC has another specific surface marker Siglec-H. We decided not to use Siglec-H, as antibody binding of this molecule represses the function of pDC to produce IFN&#945;. 11

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			<td>gibco by life technologies</td>
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			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1x Hank&#8217;s Balanced Salt Solution&#160;</td>
			<td>gibco by life technologies</td>
			<td>14175-079</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MACS BSA Stock Solution</td>
			<td>Miltenyi Biotec</td>
			<td>130-091-376</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">MgCl2</td>
			<td>SIGMA</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNase I</td>
			<td>SIGMA</td>
			<td>D4527</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Red blood cell (RBC) lysis buffer&#160;</td>
			<td>eBioscience</td>
			<td>00-4300-54</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Density gradient medium</td>
			<td>GE Healthcare</td>
			<td>17-1440-02</td>
			<td>Ficoll-Paque Plus&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-mouse CD19-PE</td>
			<td>BD Pharmingen</td>
			<td>553786</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-mouse CD11c-PE</td>
			<td>eBioscience</td>
			<td>12-0114-82</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-mouse CD11b-APC-CY7</td>
			<td>BD Pharmingen</td>
			<td>557657</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-mouse PDCA-1-FITC</td>
			<td>eBioscience</td>
			<td>11-3172-81</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-mouse B220-V500&#160;</td>
			<td>BD Pharmingen</td>
			<td>561226</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DAPI</td>
			<td>invitrogen</td>
			<td>D3571</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plasmacytoid Dendritic Cell Isolation Kit II, mouse</td>
			<td>Miltenyi Biotec</td>
			<td>130-092-786</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BD FACSAria I flow cytometer&#160;</td>
			<td>BD Biosciences</td>
			<td>643178</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BD FACS Diva version 6</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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 aimed to enrich bone marrow pDC with high purity, and without the influence of other cell types, from MRL/lpr lupus-prone mice of both young and old age to study the functional changes of pDC regarding their ability to produce IFN&#945;. The first purification strategy used was MCS, which, as Figure 1 shows, led to only 7.75% purity after the enrichment. Compared to MCS, FACS enriched pDC with purity as high as 96.4%. To ensure high purity, a step-by-step gating strategy was performed. As shown in Figure 2, mononuclear cells were gated and separated from debris by forward scatter (FSC) and side scatter (SSC) parameters. Using FSC-width (FSC-W) vs. FSC-height (FSC-H) and SSC-W vs. SSC-H, single cells were gated to avoid the false positive signals produced by aggregates. Live single cells were then gated as the DAPI- population (DAPI+ cells are dead or apoptotic). From live single cells, CD11c+CD11b- population was gated, from which B220+PDCA-1+ population was further gated as pDC. The sorted pDC were not only of high purity but also functionally normal. They had the ability to produce IFN&#945; in vitro upon CpG stimulation as shown in Figure 3 (Re-print with permission from our previous publication17.
<img alt="Figure 1" src="/files/ftp_upload/54641/54641fig1.jpg" /> 
Figure 1. The Purity of pDC Sorted through either MCS and FACS Method. Representative flow cytometry figures showing the percentage of pDC (CD11c+CD11b-B220+PDCA-1+) in sorted cells by MCS (top) and FACS (bottom), respectively. The CD11c+CD11b- population was gated from total live cells (left), and then pDC as B220+PDCA-1+ cells were gated within the CD11c+CD11b- population (right). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54641/54641fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54641/54641fig2.jpg" /> 
Figure 2. Gating Strategy for pDC Enrichment by FACS. A step-by-step gating strategy in the following order: mononuclear cells to single cells FSC, to single cells SSC, to DAPI- live cells, to CD11c+CD11b-, and to B220+PDCA-1+ as live pDC. pDC in total single cells is 2.95 &#177; 1.42 %; sorted pDC number is 0.88 &#177; 0.28 x 105 per mouse with a recovery rate around 50%. (n= 12 MRL/lpr mice, data are shown as mean &#177; SD in 95% CI of the mean).&#160; <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54641/54641fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54641/54641fig3.jpg" /> 
Figure 3. IFN&#945; Production by FACS-sorted pDC. Sorted bone marrow pDC were treated with class A CpG (ODN1585; 5 &#956;M) for 6 hr in vitro. In the figure, &#34;y&#34; stands for younger, 6-week-old mice; &#34;o&#34; stands for older, 16-week-old mice. MRL stands for MRL mice as healthy control mice; lpr stands for MRL/lpr mice as lupus-prone mice. The concentration of IFN&#945; in the culture supernatant was measured with ELISA. *P&#60; 0.05, **P&#60; 0.01, ***P&#60; 0.001, one-way ANOVA. Data are shown as mean &#177; standard error of the mean (n= 3 mice per group). This figure is reproduced from our publication in Journal of Immunology with permission. 17 <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54641/54641fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow at JoVE.com

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

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 ...

fluorescence-activated-cell-sorting-for-purification-plasmacytoid

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

12.1K Views.  Virginia Polytechnic Institute and State University. 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.

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

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS)

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known phenotype.  Other bulk methods of purification include panning, complement depletion and magnetic bead separation.  However, FACS has several advantages over other available methods.  FACS is the preferred method when very high purity of the desired population is required, when the target cell population expresses a very low level of the identifying marker or when cell populations require separation based on differential marker density.  In addition, FACS is the only available purification technique to isolate cells based on internal staining or intracellular protein expression, such as a genetically modified fluorescent protein marker.  FACS allows the purification of individual cells based on size, granularity and fluorescence.  In order to purify cells of interest, they are first stained with fluorescently-tagged monoclonal antibodies (mAb), which recognize specific surface markers on the desired cell population (1).  Negative selection of unstained cells is also possible.  FACS purification requires a flow cytometer with sorting capacity and the appropriate software.  For FACS, cells in suspension are passed as a stream in droplets with each containing a single cell in front of a laser.  The fluorescence detection system detects cells of interest based on predetermined fluorescent parameters of the cells.  The instrument applies a charge to the droplet containing a cell of interest and an electrostatic deflection system facilitates collection of the charged droplets into appropriate collection tubes (2).  The success of staining and thereby sorting depends largely on the selection of the identifying markers and the choice of mAb.  Sorting parameters can be adjusted depending on the requirement of purity and yield.  Although FACS requires specialized equipment and personnel training, it is the method of choice for isolation of highly purified cell populations.

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting FACS

For many scientific studies requiring a biological and chemical analysis of cell populations the cells must be in a high state of purity. Fluorescence activated cell sorting (FACS) is a superior method in which to obtain pure cell populations.

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known ...

Generation and Labeling of Murine Bone Marrow-derived Dendritic Cells with Qdot Nanocrystals for Tracking Studies

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone marrow, spleen, lymph nodes and Peyer's patches 1-3. DCs present in peripheral tissues sample the organism for the presence of antigens, which they take up, process and present in their surface in the context of major histocompatibility molecules (MHC). Then, antigen-loaded DCs migrate to immunological organs where they present the processed antigen to T lymphocytes triggering specific immune responses. One way to evaluate the migratory capabilities of DCs is to label them with fluorescent dyes 4.
Herewith we demonstrate the use of Qdot fluorescent nanocrystals to label murine bone marrow-derived DC. The advantage of this labeling is that Qdot nanocrystals possess stable and long lasting fluorescence that make them ideal for detecting labeled cells in recovered tissues. To accomplish this, first cells will be recovered from murine bone marrows and cultured for 8 days in the presence of granulocyte macrophage-colony stimulating factor in order to induce DC differentiation. These cells will be then labeled with fluorescent Qdots by short in vitro incubation. Stained cells can be visualized with a fluorescent microscopy. Cells can be injected into experimental animals at this point or can be into mature cells upon in vitro incubation with inflammatory stimuli. In our hands, DC maturation did not determine loss of fluorescent signal nor does Qdot staining affect the biological properties of DCs. Upon injection, these cells can be identified in immune organs by fluorescent microscopy following typical dissection and fixation procedures.

Dendritic cells uptake antigens and migrate towards immune organs to present processed antigens to T cells. Qdot nanocrystal labeling provides a long-lasting and stable fluorescent signal. This allows tracking of dendritic cells to different organs by fluorescent microscopy.

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone ...

Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice to colitis. If the gene-of-interest is expressed in both bone marrow derived cells and non-bone marrow derived cells of the host; however, it is possible to differentiate the role of a gene of interest in bone marrow derived cells and non- bone marrow derived cells by bone marrow transplantation technique. To change the bone marrow derived cell genotype of mice, the original bone marrow of recipient mice were destroyed by irradiation and then replaced by new donor bone marrow of different genotype. When wild-type mice donor bone marrow was transplanted to knockout mice, we could generate knockout mice with wild-type gene expression in bone marrow derived cells. Alternatively, when knockout mice donor bone marrow was transplanted to wild-type recipient mice, wild-type mice without gene-of-interest expressing from bone marrow derived cells were produced. However, bone marrow transplantation may not be 100% complete. Therefore, we utilized cluster of differentiation (CD) molecules (CD45.1 and CD45.2) as markers of donor and recipient cells to track the proportion of donor bone marrow derived cells in recipient mice and success of bone marrow transplantation. Wild-type mice with CD45.1 genotype and knockout mice with CD45.2 genotype were used. After irradiation of recipient mice, the donor bone marrow cells of different genotypes were infused into the recipient mice. When the new bone marrow regenerated to take over its immunity, the mice were challenged by chemical agent (dextran sodium sulfate, DSS 5%) to induce colitis. Here we also showed the method to induce colitis in mice and evaluate the role of the gene of interest expressed from bone-marrow derived cells. If the gene-of-interest from the bone derived cells plays an important role in the development of the disease (such as colitis), the phenotype of the recipient mice with bone marrow transplantation can be significantly altered. At the end of colitis experiments, the bone marrow derived cells in blood and bone marrow were labeled with antibodies against CD45.1 and CD45.2 and their quantitative ratio of existence could be used to evaluate the success of bone marrow transplantation by flow cytometry. Successful bone marrow transplantation should show a vast majority of donor genotype (in term of CD molecule marker) over recipient genotype in both the bone marrow and blood of recipient mice.

Bone marrow transplantation provides a way to change the genotype of the bone marrow derived cells. If the gene of interest is expressed in both bone marrow derived cells and non-bone marrow derived cells, bone marrow transplantation can change the bone marrow derived cells to a different genotype without changing the non-bone marrow derived cell genotype.

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice ...

Isolation, Purification and Labeling of Mouse Bone Marrow Neutrophils for Functional Studies and Adoptive Transfer Experiments

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or pathogenic effects depending on the inflammatory milieu. Nonetheless, despite the indispensable role of neutrophils in immunity, detailed understanding of the molecular factors that mediate neutrophils' effector and immunopathogenic effects in different infectious diseases and inflammatory conditions is still lacking, partly because of their short half life, the difficulties with handling of these cells and the lack of reliable experimental protocols for obtaining sufficient numbers of neutrophils for downstream functional studies and adoptive transfer experiments. Therefore, simple, fast, economical and reliable methods are highly desirable for harvesting sufficient numbers of mouse neutrophils for assessing functions such as phagocytosis, killing, cytokine production, degranulation and trafficking. To that end, we present a reproducible density gradient centrifugation-based protocol, which can be adapted in any laboratory to isolate large numbers of neutrophils from the bone marrow of mice with high purity and viability. Moreover, we present a simple protocol that uses CellTracker dyes to label the isolated neutrophils, which can then be adoptively transferred into recipient mice and tracked in several tissues for at least 4 hr post-transfer using flow cytometry. Using this approach, differential labeling of neutrophils from wild-type and gene-deficient mice with different CellTracker dyes can be successfully employed to perform competitive repopulation studies for evaluating the direct role of specific genes in trafficking of neutrophils from the blood into target tissues in vivo.

We describe a protocol for isolation and purification of neutrophils from mouse bone marrow by density gradient centrifugation and for neutrophil labeling using CellTracker dyes. This represents a simple, fast, reproducible and economical method for obtaining large numbers of neutrophils for downstream functional studies or adoptive transfer and tracking experiments.

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or ...

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer's Patches Using Adoptive Transfer of Hematopoietic Progenitors

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal Peyer&#39;s patch (PP). Common dendritic cell progenitors (CDPs, lin- c-kitlo CD115+ Flt3+) were purified from the bone marrow of C57BL6 mice by FACS and transferred to recipient mice that lack a significant pDC population in PP; in this case, Ifnar-/- mice were used as the transfer recipients. In some mice, overexpression of the dendritic cell growth factor Flt3 ligand (Flt3L) was enforced prior to adoptive transfer of CDPs, using hydrodynamic gene transfer (HGT) of Flt3L-encoding plasmid. Flt3L overexpression expands DC populations originating from transferred (or endogenous) hematopoietic progenitors. At 7-10 days after progenitor transfer, pDCs that arise from the adoptively transferred progenitors were distinguished from recipient cells on the basis of CD45 marker expression, with pDCs from transferred CDPs being CD45.1+ and recipients being CD45.2+. The ability of transferred CDPs to contribute to the pDC population in PP and to respond to Flt3L was evaluated by flow cytometry of PP single cell suspensions from recipient mice. This method may be used to test whether other progenitor populations are capable of generating PP pDCs. In addition, this approach could be used to examine the role of factors that are predicted to affect pDC development in PP, by transferring progenitor subsets with an appropriate knockdown, knockout or overexpression of the putative developmental factor and/or by manipulating circulating cytokines via HGT. This method may also allow analysis of how PP pDCs affect the frequency or function of other immune subsets in PPs. A unique feature of this method is the use of Ifnar-/- mice, which show severely depleted PP pDCs relative to wild type animals, thus allowing reconstitution of PP pDCs in the absence of confounding effects from lethal irradiation.

This protocol describes experimental procedures to assess the differentiation of plasmacytoid dendritic cells in Peyer&#8217;s patch from common dendritic cell progenitors, using techniques involving FACS-mediated cell isolation, hydrodynamic gene transfer, and flow analysis of immune subsets in Peyer&#8217;s patch.

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal ...

Tracking Mouse Bone Marrow Monocytes In Vivo

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal environment. Biological activities of immune cells mainly rely on their motility capacities. Blood monocytes have short half-life in the bloodstream; they originate in the bone marrow and are constitutively released from it. In inflammatory condition, this process is enhanced, leading to blood monocytosis and subsequent infiltration of the peripheral inflammatory tissues. Identifying the biomechanical events controlling monocyte trafficking from the bone marrow towards the vascular network is an important step to understand monocyte physiopathological relevance. We performed in vivo time-lapse imaging by two-photon microscopy of the skull bone marrow of the Csf1r-Gal4VP16/UAS-ECFP (MacBlue) mouse. The MacBlue mouse expresses the fluorescent reporters enhanced cyan fluorescent protein (ECFP) under the control of a myeloid specific promoter 1, in combination with vascular network labelling. We describe how this approach enables the tracking of individual medullar monocytes in real time to further quantify the migratory behaviour within the bone marrow parenchyma and the vasculature, as well as cell-to-cell interactions. This approach provides novel insights into the biology of the bone marrow monocyte subsets and allows to further address how these cells can be influenced in specific pathological conditions.

Tracking Mouse Bone Marrow Monocytes In Vivo

Monocytes are key regulators of innate immunity and play a critical role in the renewal of the peripheral mononuclear phagocytic system and in case of inflammation. This manuscript describes the procedure of real time imaging of the mouse calvaria bone marrow to study the monocyte mobilisation mechanism.

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal ...

In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System

Plasmacytoid dendritic cells (pDCs) are powerful type I interferon (IFN-I) producing cells that are activated in response to infection or during inflammatory responses. Unfortunately, study of pDC function is hindered by their low frequency in lymphoid organs, and existing methods for in vitro DC generation predominantly favor the production of cDCs over pDCs. Here we present a unique approach to efficiently generate pDCs from common lymphoid progenitors (CLPs) in vitro. Specifically, the protocol described details how to purify CLPs from bone marrow and generate pDCs by coculturing with &#947;-irradiated AC-6 feeder cells in the presence of Flt3 ligand. A unique characteristic of this culture system is that the CLPs migrate underneath the AC-6 cells and become cobblestone area-forming cells, a critical step for expanding pDCs. Morphologically distinct DCs, namely pDCs and cDCs, were generated after approximately 2 weeks with a composition of 70-90% pDCs under optimal conditions. Typically, the number of pDCs generated by this method is roughly 100-fold of the number of CLPs seeded. Therefore, this is a novel system with which to robustly generate the large numbers of pDCs required to facilitate further studies into the development and function of these cells.

In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System

In this study we present an in vitro culture system that can efficiently generate pDCs by co-culturing common lymphoid progenitors with AC-6 feeder cells in the presence of Flt3 ligand.

Plasmacytoid dendritic cells (pDCs) are powerful type I interferon (IFN-I) producing cells that are activated in response to infection or during ...

Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. However, they are difficult to isolate in sufficient numbers to study them in detail, therefore, in vitro models have been developed. In vitro cultures of bone marrow-derived macrophages and dendritic cells are well-established and valuable methods for immunological studies. Here, a method for culturing and identifying both DCs and macrophages from a single culture of primary mouse bone marrow cells using the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF) is described. This protocol is based on the established procedure first developed by Lutz et al. in 1999 for bone marrow-derived DCs. The culture is heterogeneous, and MHCII and fluoresceinated hyaluronan (FL-HA) are used to distinguish macrophages from immature and mature DCs. These GM-CSF derived macrophages provide a convenient source of in vitro derived macrophages that closely resemble alveolar macrophages in both phenotype and function.

Bone marrow cells cultured with granulocyte macrophage colony stimulating factor (GM-CSF) generate a heterogeneous culture containing macrophages and dendritic cells (DCs). This method highlights using MHCII and hyaluronan (HA) binding to differentiate macrophages from the DCs in the GM-CSF culture. Macrophages in this culture have many similarities to alveolar macrophages.

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. ...

Generation of Large Numbers of Myeloid Progenitors and Dendritic Cell Precursors from Murine Bone Marrow Using a Novel Cell Sorting Strategy

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have recently been recognized to be more heterogeneous than previously appreciated. These cultures routinely contain moDC as well monocyte-derived macrophages (moMac), and even some less developed cells such as monocytes. The goal of this protocol is to provide a consistent method for identification and separation of the many cell types present in these cultures as they develop, so that their specific functions may be further investigated. The sorting strategy presented here separates cells first into four populations based on expression of Ly6C and CD115, both of which are expressed transiently by cells as they develop in GM-CSF-driven culture. These four populations include Common myeloid progenitors or CMP (Ly6C-, CD115-), granulocyte/macrophage progenitors or GMP (Ly6C+, CD115-), monocytes (Ly6C+, CD115+), and monocyte-derived macrophages or moMac (Ly6C-, CD115+). CD11c is also added to the sorting strategy to distinguish two populations within the Ly6C-, CD115- population: CMP (CD11c-) and moDC (CD11c+). Finally, two populations may be further distinguished within the Ly6C-, CD115+ population based on the level of MHC class II expression. MoMacs express lower levels of MHC class II, while a monocyte-derived DC precursor (moDP) expresses higher MHC class II. This method allows for the reliable isolation of several developmentally distinct populations in numbers sufficient for a variety of functional and developmental analyses. We highlight one such functional readout, the differential responses of these cell types to stimulation with Pathogen-Associated Molecular Patterns (PAMPs).

Here we provide a method for identifying and isolating large numbers of GM-CSF driven myeloid cells using high speed cell sorting. Five distinct populations (Common myeloid progenitors, granulocyte/macrophage progenitors, monocytes, monocyte-derived macrophages, and monocyte-derived DCs) can be identified based on Ly6C and CD115 expression.

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have ...

An In Vivo Mouse Model to Measure Na&#239;ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played by T cells in an immune response. This protocol describes the in vitro differentiation of bone marrow (BM) progenitors to obtain granulocyte macrophage colony-stimulating factor (GM-CSF) derived-dendritic cells (DCs). The protocol also describes the adoptive transfer of ovalbumin peptide (OVAp)-loaded GM-CSF-derived DCs and na&#239;ve CD4 T cells from OTII transgenic mice in order to analyze the in vivo activation, proliferation, and Th1 differentiation of the transferred CD4 T cells. This protocol circumvents the limitation of purely in vivo methods imposed by the inability to specifically manipulate or select the studied cell population. Moreover, this protocol allows studies in an in vivo environment, thus avoiding alterations to functional factors that may occur in vitro and including the influence of cell types and other factors only found in intact organs. The protocol is a useful tool for generating changes in DCs and T cells that modify adaptive immune responses, potentially providing important results to understand the origin or development of numerous immune associated diseases.

Here, we present a protocol for the in vivo determination of na&#239;ve CD4 T cell (T cell) activation, proliferation, and Th1 differentiation induced by GM-CSF bone marrow (BM)-derived dendritic cells (DCs). In addition, this protocol describes BM and T-cell isolation, DC generation, and DC and T-cell adoptive transfer.

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played ...