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>

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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

<table><tbody><tr><td>RPMI 1640</td><td>gibco by life technologies</td><td>11875-093</td><td></td></tr><tr><td>Fetal bovine serum</td><td>HyClone</td><td>SH30396.03</td><td></td></tr><tr><td>Sodium pyruvate</td><td>gibco by life technologies</td><td>11360-070</td><td></td></tr><tr><td>MEM non-essential amino acids</td><td>gibco by life technologies</td><td>11140-050</td><td></td></tr><tr><td>HEPES</td><td>gibco by life technologies</td><td>15630-080</td><td></td></tr><tr><td>2-mercaptoethanol</td><td>gibco by life technologies</td><td>21985-023</td><td></td></tr><tr><td>L-glutamine&amp;nbsp;</td><td>gibco by life technologies</td><td>25030-164</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>gibco by life technologies</td><td>15140-122</td><td></td></tr><tr><td>1x Hank&amp;rsquo;s Balanced Salt Solution&amp;nbsp;</td><td>gibco by life technologies</td><td>14175-079</td><td></td></tr><tr><td>MACS BSA Stock Solution</td><td>Miltenyi Biotec</td><td>130-091-376</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>SIGMA</td><td>M8266</td><td></td></tr><tr><td>DNase I</td><td>SIGMA</td><td>D4527</td><td></td></tr><tr><td>Red blood cell (RBC) lysis buffer&amp;nbsp;</td><td>eBioscience</td><td>00-4300-54</td><td></td></tr><tr><td>Density gradient medium</td><td>GE Healthcare</td><td>17-1440-02</td><td>Ficoll-Paque Plus&amp;nbsp;</td></tr><tr><td>anti-mouse CD19-PE</td><td>BD Pharmingen</td><td>553786</td><td></td></tr><tr><td>anti-mouse CD11c-PE</td><td>eBioscience</td><td>12-0114-82</td><td></td></tr><tr><td>anti-mouse CD11b-APC-CY7</td><td>BD Pharmingen</td><td>557657</td><td></td></tr><tr><td>anti-mouse PDCA-1-FITC</td><td>eBioscience</td><td>11-3172-81</td><td></td></tr><tr><td>anti-mouse B220-V500&amp;nbsp;</td><td>BD Pharmingen</td><td>561226</td><td></td></tr><tr><td>DAPI</td><td>invitrogen</td><td>D3571</td><td></td></tr><tr><td>Plasmacytoid Dendritic Cell Isolation Kit II, mouse</td><td>Miltenyi Biotec</td><td>130-092-786</td><td></td></tr><tr><td>BD FACSAria I flow cytometer&amp;nbsp;</td><td>BD Biosciences</td><td>643178</td><td></td></tr><tr><td>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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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

Universidad San Sebastian

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 ...

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 ...

Preparation of Myeloid Derived Suppressor Cells (MDSC) from Naive and Pancreatic Tumor-bearing Mice using Flow Cytometry and Automated Magnetic Activated Cell Sorting (AutoMACS)

MDSC are a heterogeneous population of immature macrophages, dendritic cells and granulocytes that accumulate in lymphoid organs in pathological conditions including parasitic infection, inflammation, traumatic stress, graft-versus-host disease, diabetes and cancer1-7. In mice, MDSC express Mac-1 (CD11b) and Gr-1 (Ly6G and Ly6C) surface antigens7. It is important to note that MDSC are well studied in various tumor-bearing hosts where they are significantly expanded and suppress anti-tumor immune responses compared to na&iuml;ve counterparts7-10. However, depending on the pathological condition, there are different subpopulations of MDSC with distinct mechanisms and targets of suppression11,12. Therefore, effective methods to isolate viable MDSC populations are important in elucidating their different molecular mechanisms of suppression in vitro and in vivo.
Recently, the Ghansah group has reported the expansion of MDSC in a murine pancreatic cancer model. Our tumor-bearing MDSC display a loss of homeostasis and increased suppressive function compared to na&iuml;ve MDSC 13. MDSC percentages are significantly less in lymphoid compartments of na&iuml;ve vs. tumor-bearing mice. This is a major caveat, which often hinders accurate comparative analyses of these MDSC. Therefore, enriching Gr-1+ leukocytes from na&iuml;ve mice prior to Fluorescence Activated Cell Sorting (FACS) enhances purity, viability and significantly reduces sort time. However, enrichment of Gr-1+ leukocytes from tumor-bearing mice is optional as these are in abundance for quick FACS sorting. Therefore, in this protocol, we describe a highly efficient method of immunophenotyping MDSC and enriching Gr-1+ leukocytes from spleens of na&iuml;ve mice for sorting MDSC in a timely manner. Immunocompetent C57BL/6 mice are inoculated with murine Panc02 cells subcutaneously whereas na&iuml;ve mice receive 1XPBS. Approximately 30 days post inoculation; spleens are harvested and processed into single-cell suspensions using a cell dissociation sieve. Splenocytes are then Red Blood Cell (RBC) lysed and an aliquot of these leukocytes are stained using fluorochrome-conjugated antibodies against Mac-1 and Gr-1 to immunophenotype MDSC percentages using Flow Cytometry. In a parallel experiment, whole leukocytes from na&iuml;ve mice are stained with fluorescent-conjugated Gr-1 antibodies, incubated with PE-MicroBeads and positively selected using an automated Magnetic Activated Cell Sorting (autoMACS) Pro Separator. Next, an aliquot of Gr-1+ leukocytes are stained with Mac-1 antibodies to identify the increase in MDSC percentages using Flow Cytometry. Now, these Gr1+ enriched leukocytes are ready for FACS sorting of MDSC to be used in comparative analyses (na&iuml;ve vs. tumor- bearing) in in vivo and in vitro assays.

Preparation of Myeloid Derived Suppressor Cells MDSC from Naive and Pancreatic Tumor-bearing Mice using Flow Cytometry and Automated Magnetic Activated Cell Sorting AutoMACS

This is a rapid and comprehensive method of immunophenotyping Myeloid Derived Suppressor Cells (MDSC) and enriching Gr-1+ leukocytes from mouse spleens. This method uses flow cytometry and AutoMACS Cell Sorting to enrich for viable Gr-1+ leukocytes prior to FACS sorting of MDSC for use in vivo and in vitro assays.

MDSC are a heterogeneous population of immature macrophages, dendritic cells and granulocytes that accumulate in lymphoid organs in pathological ...

Medicine

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 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 ...

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 ...

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 ...

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.

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 ...

Economical and Efficient Protocol for Isolating and Culturing Bone Marrow-derived Dendritic Cells from Mice

The demand for dendritic cells (DCs) is gradually increasing as immunology research advances. However, DCs are rare in all tissues. The traditional method for isolating DCs primarily involves inducing bone marrow (BM) differentiation into DCs by injecting large doses (&#62;10 ng/mL) of granulocyte-macrophage colony-stimulating factor/interleukin-4 (GM-CSF/IL-4), making the procedure complex and expensive. In this protocol, using all BM cells cultured in 10 ng/mL GM-CSF/IL-4 medium, after 3-4 half-culture exchanges, up to 2.7 x 107 CD11c+ cells&#160;(DCs) per mouse (two femurs) were harvested with a purity of 80%-95%. After 10 days in culture, the expression of CD11c, CD80, and MHC II increased, whereas the number of cells decreased. The number of cells peaked after 7 days of culture. Moreover, this method only took 10 min to harvest all bone marrow cells, and a high number of DCs were obtained after 1 week of culture.

Here, we present an economical and efficient method to isolate and generate high-purity bone marrow-derived dendritic cells from mice after 7 days of culture with 10 ng/mL GM-CSF/IL-4.

The demand for dendritic cells (DCs) is gradually increasing as immunology research advances. However, DCs are rare in all tissues. The traditional method ...

In Vitro Generation of Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors

Source: Chang, S., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/53211">In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System.</a> J. Vis. Exp. (2015)
This video demonstrates an in vitro assay to generate plasmacytoid dendritic cells (pDCs) from common lymphoid progenitor cells (CLPs). Upon co-culturing purified CLPs with &#947;-irradiated feeder cells in the presence of Fms-like tyrosine kinase-3 ligand (FL), the CLPs differentiate into non-adherent pDCs, the presence of which is confirmed using flow cytometry.

Source: Chang, S., et al. In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System. J. ...

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

Summary

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Chapters in this video

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

Isolation of Mouse Lung Dendritic Cells

Preparation of Myeloid Derived Suppressor Cells (MDSC) from Naive and Pancreatic Tumor-bearing Mice using Flow Cytometry and Automated Magnetic Activated Cell Sorting (AutoMACS)

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

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

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

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

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

Economical and Efficient Protocol for Isolating and Culturing Bone Marrow-derived Dendritic Cells from Mice

In Vitro Generation of Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors