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.

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...

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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.

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 strate...

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 ...

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.

Research

JoVE Journal

Immunology and Infection

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

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