مضان تنشيط الفرز الخليوي لتنقية الخلايا الجذعية بلازماوية الشكل من الفأر نخاع العظم
Summary
نفيدكم بروتوكول باستخدام مضان تنشيط الخلايا الفرز لعزل الخلايا الجذعية بلازماوية الشكل (PDC) مع نقاء عالية من نخاع العظام من الفئران المعرضة للمرض الذئبة للدراسات وظيفية من الحزب الديمقراطي المسيحي.
Abstract
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.
Introduction
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 IFNα. 11
Protocol
Representative Results
Discussion
The protocol described in this manuscript is for high purity enrichment of live pDC that retain the ability to produce IFNα. 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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| RPMI 1640 | gibco by life technologies | 11875-093 | |
| Fetal bovine serum | HyClone | SH30396.03 | |
| Sodium pyruvate | gibco by life technologies | 11360-070 | |
| MEM non-essential amino acids | gibco by life technologies | 11140-050 | |
| HEPES | gibco by life technologies | 15630-080 | |
| 2-mercaptoethanol | gibco by life technologies | 21985-023 | |
| L-glutamine | gibco by life technologies | 25030-164 | |
| Penicillin-Streptomycin | gibco by life technologies | 15140-122 | |
| 1x Hank’s Balanced Salt Solution | gibco by life technologies | 14175-079 | |
| MACS BSA Stock Solution | Miltenyi Biotec | 130-091-376 | |
| MgCl2 | SIGMA | M8266 | |
| DNase I | SIGMA | D4527 | |
| Red blood cell (RBC) lysis buffer | eBioscience | 00-4300-54 | |
| Density gradient medium | GE Healthcare | 17-1440-02 | Ficoll-Paque Plus |
| anti-mouse CD19-PE | BD Pharmingen | 553786 | |
| anti-mouse CD11c-PE | eBioscience | 12-0114-82 | |
| anti-mouse CD11b-APC-CY7 | BD Pharmingen | 557657 | |
| anti-mouse PDCA-1-FITC | eBioscience | 11-3172-81 | |
| anti-mouse B220-V500 | BD Pharmingen | 561226 | |
| DAPI | invitrogen | D3571 | |
| Plasmacytoid Dendritic Cell Isolation Kit II, mouse | Miltenyi Biotec | 130-092-786 | |
| BD FACSAria I flow cytometer | BD Biosciences | 643178 | |
| BD FACS Diva version 6 | BD Biosciences |
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