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.

注：MRL / MP-のFas のlpr狼瘡傾向マウスを繁殖させたとバージニア工科大学（動物福祉保証番号での施設内動物管理使用委員会（IACUC）の要件以下の特定病原体フリーの施設で維持した（MRL / LPR）： A3208-01）。この研究は、米国立衛生研究所の実験動物の管理と使用に関するガイドの推奨事項に厳密に従って行きました。全ての動物実験は、IACUCプロトコル番号12から062で行いました。 <p class="jove_title...

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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.

我々は、IFNαを産生する能力についてのpDCの機能変化を研究するために老いも若き両方年齢のMRL / LPRの狼瘡傾向マウスから、高純度の骨髄のpDCを豊かにすることを目的とした、および他の細胞型の影響を受けません。使用された最初の精製戦略は、 図1に示すように、濃縮した後にのみ7.75パーセントの純度につながった、MCS、でした。 MCSと比較して、FACS?...

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

私たちは、PDCの機能研究のための狼瘡傾向マウスの骨髄から高純度の形質細胞様樹状細胞（pDC）を単離するために蛍光活性化セルソーティングを使用してプロトコルを報告しています。

マウスの骨髄から形質細胞様樹状細胞の精製のために、蛍光活性化細胞選別

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

蛍光活性化細胞選別（FACS）によって特定の細胞集団の精製

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

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

追跡調査のためのQdot ®二ナノとマウス骨髄由来樹状細胞の世代とラベリング

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

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

マウスの腸からの樹状細胞およびマクロファージの単離とキャラクタリゼーション

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

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

骨髄移植によるマウス大腸炎における免疫および非免疫細胞からの遺伝子発現の機能的役割を差別化

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

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

機能研究のためのマウス骨髄好中球と養子移入実験の分離、精製とラベリング

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

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

造血前駆細胞の養子移入を使用して、パイエル板におけるマウス形質細胞様樹状細胞の開発を評価

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

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

AC-6フィーダシステムを使用してリンパ球系共通前駆細胞からマウス形質細胞様樹状細胞のインビトロ生成に

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

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

マウスの骨髄からGM-CSF由来肺胞のようなマクロファージや樹状細胞の生成および同定

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

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

大量の骨髄前駆細胞と新規セルソーティング戦略を使用してマウスの骨髄から樹状細胞前駆体の生成

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