Name: fluorescence-activated cell sorting for purification of plasmacytoid dendritic cells from the mouse bone marrow
Uploaded: 2016-11-04T14:00:00
Description: Watch this Scientific Journal Video about Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow at JoVE.com

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.

OBS: MRL / Mp-Fas lpr (MRL / lpr) lupus-benägna möss avlades och hölls i ett visst patogen-fri anläggning enligt kraven i Institutional Animal Care och användning kommittén (IACUC) vid Virginia Tech (djurskydds Assurance Antal: A3208-01). Denna studie genomfördes i strikt överensstämmelse med rekommendationerna i Guide för skötsel och användning av försöksdjur i National Institutes of Health. Alla djurförsök genomfördes under IACUC protokollet # 12-062. 1. Cell Culture Medium och Sorter...

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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. 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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

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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.

Vi syftar till att berika benmärg pDC med hög renhet, och utan påverkan av andra celltyper, från MRL / lpr lupus-benägna möss av både unga och gamla ålder för att studera de funktionella förändringar PDC om deras förmåga att producera IFNa. Den första reningsstrategi som användes var MCS, som, såsom figur 1 visar, ledde till endast 7,75% renhet efter anrikning. Jämfört med MCS, FACS berikat pDC med renhet så hög som 96,4%. För att säkerställa hög ...

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

Vi rapporterar ett protokoll med användning av fluorescensaktiverad cellsortering för att isolera plasmacytoid dendritiska celler (PDC) med hög renhet från benmärgen hos lupus-benägna möss för funktionella studier av pDC.

Fluorescensaktiverad cellsortering för Rening av Plasmacytoid dendritiska celler från benmärg från mus

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

Research

JoVE Journal

Immunology and Infection

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

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting FACS

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

Skilja funktionella roller av genuttryck från immuna och icke-immuna celler i mus Kolit av benmärgstransplantation

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 ...

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

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

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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. ...

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Generation av stort antal myeloida och dendritiska cellen prekursorer från murina benmärg med en roman Cell sortering strategi

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