Name: Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow (Video) | JoVE Journal
Uploaded: 2016-11-04T14:00:00.000+00:00
Duration: 8 min 1 s
Description: Learn fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells from lupus-prone mouse bone marrow with high purity. This method enables precise functional studies of immune cell populations.

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5 minutes

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><li>Bonner, W. A., Hulett, H. R., Sweet, R. G., Herzenberg, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fluorescence+activated+cell+sorting%5BTitle%5D)+AND+%22Rev+Sci+Instrum%22%5BJournal%5D)">Fluorescence activated cell sorting.</a> Rev Sci Instrum. 43 (3), 404-409 (1972).</li>
<li>Herzenberg, L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+history+and+future+of+the+fluorescence+activated+cell+sorter+and+flow+cytometry%3A+a+view+from+Stanford%5BTitle%5D)+AND+%22Clin+Chem%22%5BJournal%5D)">The history and future of the fluorescence activated cell sorter and flow cytometry: a view from Stanford.</a> Clin Chem. 48 (10), 1819-1827 (2002).</li>
<li>Brown, M., Wittwer, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Flow+cytometry%3A+principles+and+clinical+applications+in+hematology%5BTitle%5D)+AND+%22Clin+Chem%22%5BJournal%5D)">Flow cytometry: principles and clinical applications in hematology.</a> Clin Chem. 46 (8 Pt 2), 1221-1229 (2000).</li>
<li>Laerum, O. D., Farsund, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Clinical+application+of+flow+cytometry%3A+a+review%5BTitle%5D)+AND+%22Cytometry%22%5BJournal%5D)">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/pubmed?term=((Applications+of+flow+cytometry+to+clinical+microbiology%5BTitle%5D)+AND+%22Clin+Microbiol+Rev%22%5BJournal%5D)">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/pubmed?term=((Applications+of+cell+sorting+in+biotechnology%5BTitle%5D)+AND+%22Microb+Cell+Fact%22%5BJournal%5D)">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/pubmed?term=((Analysis+of+CD95+and+CCR7+expression+on+circulating+CD4%28%2B%29+lymphocytes+revealed+disparate+immunoregulatory+potentials+in+systemic+lupus+erythematosus%5BTitle%5D)+AND+%22Saudi+J+Biol+Sci%22%5BJournal%5D)">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/pubmed?term=((Innate+and+adaptive+immunity+interact+to+quench+microbiome+flagellar+motility+in+the+gut%5BTitle%5D)+AND+%22Cell+Host+Microbe%22%5BJournal%5D)">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/pubmed?term=((High-throughput+fluorescence-based+isolation+of+live+C.+elegans+larvae%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">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/pubmed?term=((DC-SIGN+expression+on+podocytes+and+its+role+in+inflammatory+immune+response+of+lupus+nephritis%5BTitle%5D)+AND+%22Clin+Exp+Immunol%22%5BJournal%5D)">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/pubmed?term=((A+cell-surface+molecule+selectively+expressed+on+murine+natural+interferon-producing+cells+that+blocks+secretion+of+interferon-alpha%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">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/pubmed?term=((Magnetic-activated+cell+sorting+%28MCS%29+can+be+used+as+a+large-scale+method+for+establishing+zebrafish+neuronal+cell+cultures%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">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/pubmed?term=((Fluorescence-+and+magnetic-activated+cell+sorting+strategies+to+isolate+and+enrich+human+spermatogonial+stem+cells%5BTitle%5D)+AND+%22Fertil+Steril%22%5BJournal%5D)">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/pubmed?term=((Phenotypic+and+functional+change+of+cytokine-activated+neutrophils%3A+inflammatory+neutrophils+are+heterogeneous+and+enhance+adaptive+immune+responses%5BTitle%5D)+AND+%22J+Leukoc+Biol%22%5BJournal%5D)">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/pubmed?term=((The+dysregulation+of+cytokine+networks+in+systemic+lupus+erythematosus%5BTitle%5D)+AND+%22J+Interferon+Cytokine+Res%22%5BJournal%5D)">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. J., Briere, F., Trinchieri, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mouse+strain+differences+in+plasmacytoid+dendritic+cell+frequency+and+function+revealed+by+a+novel+monoclonal+antibody%5BTitle%5D)+AND+%22J+Immunol%22%5BJournal%5D)">Mouse strain differences in plasmacytoid dendritic cell frequency and function revealed by a novel monoclonal antibody.</a> J Immunol. 171 (12), 6466-6477 (2003).</li>
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</ol>

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 mi...

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 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 IFNα. 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 wa...

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 ...

The overall goal of this experiment is to isolate plasmacytoid dendritic cells, or PDC, with a high purity from the bone marrow of lupus-prone mice for the study of their function. This method can help answer key questions related to the characteristics of a particular immune cell population, such as, interferon alpha producing PDC during the late stage of lupus development. The main advantage of this technique is that one to several target populations can be enriched to a higher purity than by magnetic bead sorting.After harvesting, transfer the hind limb bones and four milliliters of complete medium, into a mortar. Add six milliliters of fresh complete medium to bring the total volume to 10 milliliters. And crack the bones gently with a pestle.Gently stir the pestle to release the bone marrow. Then, transfer all of the medium through a 70 micron strainer into a 50 milliliter conical tube on ice. Add 10 milliliters of fresh complete medium to the mortar.And filter the supernatant two more times until the bones appear white. Then, centrifuge the bone marrow cells, and re-suspend the pellet in 10 milliliters of fresh complete medium. Next, layer the cells on to five milliliters of freshly prepared density gradient medium in a 15 milliliter conical tube, taking care not to disrupt the layers.And separate the cells by centrifugation. Then, carefully remove the top eight milliliters of the solution, and collect the two milliliter buffy coat at the interphase. Add 12 milliliters of ice-cold complete medium to the isolated mononuclear cells, and cap the tube to mix by inversion.Then, pellet the cells by centrifugation. To stain the cells for FACS, re-suspend them in 100 microliters of CD1632FC blocking antibody at four degrees Celsius. After 10 minutes, wash the cells in four milliliters of ice cold sorting buffer and label the cells with 100 microliters of the appropriate cell surface staining antibody cocktail.For 15 minutes at four degree Celsius in the dark. Wash the cells in four milliliters of ice cold sorting buffer to remove any unbound fluorescence conjugated antibody. Then, re-suspend the pellet in 500 microliters of FACS loading solution and transfer the sample into a FACS tube on ice.To stain the compensation controls, aliquot one times 10 to sixth splenocytes per tube. Wash the cells with four milliliters of sorting buffer and re-suspend the pellets in 100 microliters of sorting buffer. Then, add the appropriate fluorescence conjugated antibody from the master cell surface antibody cocktail to each corresponding compensation tube and shake the tubes to mix.After 15 minutes in the dark, at four degree Celsius, wash all of the samples with sorting buffer and re-suspend the pellets in 500 microliters of ice cold sorting buffer. Except for the DAPI tube, which is re-suspended in 500 microliters of FACS loading solution. To purify the target cell population by fluorescence activated cell sorting, load collection tubes containing 500 microliters of FBS supplemented with antibiotics below the sorting valves.Next, load the unstained tube and collect 10, 000 events to set the negative gate for each fluorescent intensity. And 10, 000 events in the single stain compensation tubes to set the positive gate for each fluorescent intensity. Collect 3, 000 events from the experimental sample to set the gates of the sorted cell population.Using the fluorescence intensities as the parameters of the X and Y access graph to manually gate the target cell population. Then, sort the target cell population into the collection tube. At the end of the sorting, add up to four milliliters of ice cole complete medium to the cells.Then, cap the tube and mix the cells by inversion. Pellet the purified cells by centrifugation, aspirating all but the last 200 microliters of the supernatant. Then, re-suspend the pellet in one milliliter of ice cold complete medium and transfer the cells to a 1.5 milliliter micro centrifuge tube for a second centrifugation.Finally, aspirate all but the last 100 microliters of the supernatant and store the sorted cell population on ice until use. Magnetic activated cell sorting of bone marrow plasmacytoid dendritic cells from lupus prone mice results in only a 7.75%purity after the enrichment. FACS enriched plasmacytoid dendritic cells, however, demonstrate a purity as high as 96.4%post sorting.To ensure a high purity, the mononuclear bone marrow cells are first gated and separated from debris by forward and side scatter parameters. Using forward scatter width versus forward scatter height and side scatter width versus side scatter height, the single cells are further gated to avoid the false positive signals produced by aggregates. The live single DAPI negative cells are then gated by their CD11c positive, CD11B negative cell surface expression allowing further gating of the B220 positive, PDCA-1 positive target bone marrow plasmacytoid dendritic cell population.The sorted PDC population is not only of a high purity but also functionally normal with the ability to produce IFNalpha upon CPG stimulation in vitro. While attempting this procedure, it's important to remember to crack the bones and stir the released bone marrow gently to preserve cell viability. Following this procedure, other methods like RNA sequencing or in vitro culture and stimulation, can be performed to answer additional questions about the gene expression or function of enriched cell populations.After its development, this technique paved the way for PDC researchers to explore the role of this powerful cell in the development of lupus and lupus prone mouse models. After watching this video, you should have a good understanding of how to isolate a highly purified PDC population from the bone marrow of lupus prone mice.

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Learn fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells from lupus-prone mouse bone marrow with high purity. This method enables precise functional studies of immune cell populations.

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