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.

참고 : MRL / MP-의 Fas의 LPR 루푸스 발생하기 쉬운 쥐 사육과 버지니아 공대 (동물 복지 보증 번호에서 기관 동물 관리 및 사용위원회 (IACUC)의 요구 사항에 다음과 같은 특정 병원균이없는 시설에서 유지되었다 (MRL / LPR) : A3208-01). 이 연구는 국립 보건원 (National Institutes of Health)의 실험 동물의 관리 및 사용에 대한 가이드의 권장 사항을 엄격히 준수하여 실시 하였다. 모든 동물 실험은 IACUC 프로토콜 # 12-062 하에서 수행 하였다. 1. 세포 배양 매체 및 정렬 버퍼 <ol><li> 완전한 세포 배지 (C10)를 10 % 소 태아 혈청, 1 mM 피루브산 나트륨, 1 내지 100 % MEM 비 필수 아미노산, 10 mM의 HEPES, 55 μM 2- 머 캅토 에탄올, 2 mM L- 글루타민 및 보충 된 RPMI 1640을 사용하여 준비 100 U / ㎖ 페니실린 - 스트렙토 마이신. </li><li> 정렬 버퍼를 준비 된 10 mM HEPES가 보충 1X 행크의 균형 잡힌 염 용액 (HBSS)를 사용하여 (가득 HBSS) 2.5 ㎎ / ㎖ 소 혈청 알부민0.05 밀리미터의 MgCl 2, 0.2 U / ㎖ DNase의 I. </li></ol> 2. 마우스 해부 <ol><li> 4 ML의 C10를 포함하는 두 개의 6 cm 직경 요리를 준비합니다. 얼음에 요리를 놓습니다. </li><li> CO 2 흡입 마우스를 안락사. </li><li> 비장을 수확하고 얼음에 C10와 함께 접시에 보관합니다. <ol><li> 해부 접시에 직면하고있는 배와 마우스를 아래로 핀. 70 % 에탄올을 분무하여 몸을 소독. </li><li> 피부를 잘라 아래 목의 치골에서 복부 정중선을 따라 근육 벽에서 피부를 분리합니다. </li><li> 발목의 첫 번째 절개에서 뒷다리를 따라 피부를 잘라. </li><li> 다시 측면에있는 피부를 핀과 다이어프램하는 최초의 절개 곳에서 측면을 따라 근육 벽을 잘라. </li><li> 헤드의 우측에 근육 벽 위로 고정. </li><li> 소장 하부 위장 옆 마우스의 오른쪽에 비장을 찾아. 일의 한쪽 끝을 픽업전자 비장과 위장에서 분리. </li></ol></li><li> 대퇴골과 경골을 포함, 모두 뒷다리를 잘라하고, 가능한 한 많은 모든 근육을 제거합니다. <ol><li> 혈관을 피하기 위해 노력하고, 날카로운 가위로 발목 골반 / 고관절에서 뒷다리를 따라 근육을 잘라. </li><li> 골수 콘텐츠의 노출없이 골반 / 고관절 발목 / 발 관절 절단하여 뒷다리를 분리한다. </li><li> 반복적 면도날 긁 접합 지점에서 근육을 절단함으로써 뼈 깨끗 나머지 근육. </li></ol></li><li> 얼음에 4 ML의 C10을 포함하는 6cm 접시에 뼈를 유지합니다. 다음 마우스를 해부를 진행합니다. </li></ol> 3. 비장 격리 <ol><li> 더 레드 조각을 볼 수 없을 때까지 4 ML의 C10를 포함하는 접시의 상단에 70 μm의 셀 스트레이너에 대한 주사기 플런저 부드럽게 비장을 균질화. </li><li> 스트레이너 관통 접시에 추가 6 ML의 C10 추가ND 후 15ml의 원뿔형 튜브에 총 10 ml의 세포 현탁액을 전송. </li><li> 5 분, 4 °에 C 800 XG에서 원뿔 튜브를 원심 분리기. </li><li> 원심 분리 후, 상등액을 흡인하고 2 ㎖의 1 × 적혈구 (RBC) 용해 완충액에서 세포 펠렛을 재현 탁. 5 분 동안 실온에서 인큐베이션. </li><li> 배양 후, 5 분, 4 °에 C 800 XG에서 원뿔 튜브를 원심 분리기. </li><li> 원심 분리 후, 상등액을 흡인하고 한 ML C10에서 세포 펠렛을 재현 탁. 어떤 큰 덩어리 (죽은 세포)를 취소하고 나중에 얼음에 튜브를 유지. 주 : 많은 작은 덩어리가있는 경우, 일단 세포 현탁액을 필터링하기 위해 70 μm의 세포 여과기를 사용한다. </li><li> 자동 세포 계수기를 사용하여 트리 판 블루로 세포 수를 계산. </li></ol> 4. 골수 세포 분리 <ol><li> 박격포로 4 ML의 C10과 뼈를 전송하고 총 10 ML의 C10의 볼륨을 만들기 위해 6 ML의 C10을 추가합니다. </li><li> AP를 이용하여 모르타르에서 가볍게 뼈 균열estle. </li><li> C10에 골수를 해제 유봉으로 가볍게 저어. </li><li> 70 μm의 세포 여과기를 통해 얼음에 50 ㎖ 원뿔형 튜브에 골수를 함유하는 10 ㎖의 C10 전송. 아직 뼈가 포함 된 박격포에 10 ml의 신선한 C10을 추가합니다. 주 : 피펫 붉은 덩어리가 표시하는 경우 여과기를 통과하기 전에 골수 위아래로 수회 함유 용액. </li><li> 반복 두 번 4.4-4.2 단계를 반복합니다. 마지막 세척 후, 뼈 흰색 나타납니다. </li><li> 5 분, 4 °에 C 800 XG에 50 ML 원뿔 튜브를 원심 분리기. 한편, 실온에서 15 ml의 원추형 원심 분리 튜브에 5 ㎖를 즉시 사용 가능한 밀도 구배 매질을 제조. </li><li> 원심 분리 후, 상등액을 흡인하고 10 ㎖의 C10에서 세포 펠렛을 재현 탁. </li><li> 천천히 두 단계 들간의 인터페이스를 유지하면서 5 ml의 밀도 구배 매체의 상부에 10 ml의 세포 현탁액을 층을 포함한다. 그런 다음 30 분에 대한 1,363g에서 15 ML 원뿔 튜브를 원심 분리0 (또는 브레이크 OFF)로 설정 가속 9로 설정하고 감속와 RT (20 ° C). </li><li> 원심 분리 한 후 조심스럽게 가기 8 mL의 용액을 제거하고 새로운 15ml의 원뿔형 튜브에 인터페이스 (단핵 세포 층)의 2 mL의 버피 코트를 수집한다. </li><li> 골수 단핵 세포, 캡을 포함하는 15 ML 원뿔 관에 12 ml의 차가운 얼음 C10을 추가 한 다음, 잘 혼합 10 분, 4 °에 C 800 XG에서 튜브를 원심 분리기 여러 번 반전. </li></ol> FACS 5. 세포 표면 염색 <ol><li> 샘플 염색 <ol><li> 항 - 마우스 CD16 / 32 항체 용액 (에서 1-100 희석 HBSS - 전체 / ml의 최종 농도 5 μg의)를 준비합니다. </li><li> 단계 4.10의 원심 분리 후, 상등액을 흡인하고, 단핵 세포상의 Fc 수용체를 차단하기 위해 100 ㎕의 항 - 마우스 CD16 / 32 항체 용액 중에 세포 펠렛을 재현 탁. 10 분 동안 빙상에서 인큐베이션. </li><li> (개미 형광 - 복합 항체 혼합물을 준비내가 마우스의 CD11c-PE 1시 40분 희석, 항 - 마우스 CD11b를-APC-CY7 1:80 희석, 항 - 마우스 PDCA-1-FITC 1시 40분 희석 및 항 마우스 B220-V500 HBSS-에서 1시 40분 희석 샘플 당 100 ㎕의 최종 부피)의 제조업체에서 권장 전체. 참고 : 사용하기 전에 항체를 적정하는 것이 중요하다. </li><li> 단계 5.1.2에서 10 분 배양 한 후, 항 마우스 CD16 / 32 언 바운드 제거하기 위해 5 분, 4 ° C 800 XG에 4 ml의 얼음 차가운 HBSS - 전체와 원심 분리기를 추가합니다. </li><li> 원심 분리 후, 상등액을 흡인하고 100 ㎕의 형광 접합 된 항체 혼합물의 세포 펠렛을 재현 탁. 어둠 속에서 15 분 동안 빙상에서 인큐베이션. </li><li> 15 분 부화 후, 언 바운드 형광 복합 항체를 제거하는 5 분, 4 ° C 800 XG에 4 ml의 얼음 차가운 HBSS - 전체와 원심 분리기를 추가합니다. </li><li> FACS 로딩 솔루션 (HBSS-전체 500 NG / ㎖ DAPI)를 준비합니다. </li><li> 단계 5.1.6에서 원심 분리 후, 상층 액을 대기음 500 μ의 세포 펠렛을 재현 탁; 리터 FACS 로딩 솔루션입니다. 12 X 75mm 둥근 바닥 튜브 (FACS 튜브)에 세포 현탁액을 전송하고 1 시간 내에 정렬 될 때까지 어둠 속에서 얼음에 튜브를 유지. </li></ol></li><li> 보상 염색 <ol><li> PE, APC-CY7, FITC, V500, DAPI 또는 염색 등의 레이블 6 FACS 튜브. 1 × 10 6 세포 /를 FACS 튜브에 튜브 및에서 나누어지는 비장 세포는 5 분, 4 °에 C 800 XG에 4 ml의 HBSS - 전체 씻어. 주 : 사용 사균의 생균을 구별하는 형광 염료, DAPI를 DNA 결합. DAPI는 죽은 세포의 세포막 (그러나 살아있는 세포) 및 바인드 염색체 DNA를 통과 할 수 있습니다. </li><li> 뜨는을 대기음, 100 μl의 HBSS-전체에서 세포 펠렛을 resuspend. </li><li> 항 - 마우스 CD19-PE 1시 40분 희석, 항 - 마우스 CD11b를-APC-CY7 1:80 희석 항 마우스 PDCA -1- FITC 1시 40분 희석 또는 각각 대응 FACS 튜브 내의 다음 항체 중 하나를 추가 manufa의 권장 방지 마우스 B220-V500 1시 40분 희석cturer. 그대로 5 일 (DAPI)과 6 일 (흠) FACS 튜브를 남겨주세요. 흔들어 잘 혼합하고 15 분 동안 어둠 속에서 얼음에 모든 FACS 튜브를 품어. </li><li> 배양 후, 5 분, 4 ° C 800 XG에서 각 튜브와 원심 분리기에 FACS 튜브를 4 ml의 차가운 얼음 HBSS-전체를 추가합니다. </li><li> 원심 분리 후, 상층 액을 대기음, 500 μl의 얼음에있는 세포 펠렛을 500 ㎕의 FACS로드 용액에 재현 탁 DAPI 표시된 튜브에 펠렛을 제외하고 차가운 HBSS-전체를 재현 탁. 정렬 될 때까지 어둠 속에서 얼음에 모두 6 튜브를 유지. </li></ol></li></ol> 6. 셀 분류기에 정렬 주 : 사이토와 소프트웨어 절차를 정렬하는 동작은, 회사가 제공하는 구체적인 명령으로 표준화된다. 간단히 말해서, 우리는 20 psi에서 100 미크론 노즐, 5 사이의 설정 대상 셀 농도 사용 - ml의 당 10 만, 70 % 이상으로 효율을 조정 (즉, 충돌 30 아래에 보관%). <ol><li> 100 U / ㎖ 페니실린 - 스트렙토 마이신이 포함 된 500 μL의 FBS / 튜브 수집 FACS 튜브를 준비합니다. </li><li> 단계 5.2에서 단일 얼룩 보정 튜브를 이용한 셀 소터의 보정 파라미터를 조정한다. <ol><li> 각각의 형광 강도에 대한 부정적인 게이트를 설정 흠 튜브 만 셀을 기록합니다. </li><li> 각각의 형광 강도에 대한 긍정적 인 게이트를 설정하는 다른 단일 얼룩 보상 튜브 만 셀을 기록합니다. 주 : 소프트웨어가 자동으로 보정 파라미터를 계산합니다. </li></ol></li><li> 3,000 셀을 기록하여 정렬 된 세포 집단의 게이트를 설정하는 단계 5.1에서 하나의 샘플을 사용합니다. 수동 분명히 다른 점에서 분리 점 그룹으로 X 및 Y 축 그래프 게이트 표적 세포 집단의 파라미터 형광 강도 사용. 참고 :이 게이팅 세트는 연구자의 요구 사항에 따라 유연하고 임의이다. 연구자들은 하나 이상의 마커에 기초하여 높은 순도를 기대하는 경우, 게이트 이동높은 대응하는 형광 강도에 따라. </li><li> 컬렉션 튜브를로드하고 대상 세포 인구를 정렬 시작합니다. </li><li> 모든 샘플 튜브가 완료 될 때까지 정렬 한 후 얼음에 포집 관을 유지합니다. </li><li> 4 ml의, 모자까지 포집 관에 얼음 감기의 C10을 추가하고 혼합하는 반전. </li><li> 5 분, 4 ° C 800 XG에서 수집 튜브를 원심 분리기, 다음의 손길이 닿지 않은 세포 펠렛으로 약 200 μl를 떠나, 뜨는을 대기음. </li><li> 세포 펠렛을 재현 탁하고, 1.5 ml의 원심 분리 관에 모든 세포 현탁액을 전송하기 위해 1 ml의 얼음 추위에 C10을 추가합니다. </li><li> 원심 분리기의 손길이 닿지 않은 세포 펠렛 100 μl를 떠나는 1.5 ML의 5 분, 4 ° C 800 XG에 튜브와 뜨는을 대기음. </li><li> 사용할 때까지 얼음에 정렬 된 세포 집단을 저장합니다. </li></ol>

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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. A., Lieberman, L. A., Kis-Toth, K., Crispin, J. C., Tsokos, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dysregulation+of+cytokine+networks+in+systemic+lupus+erythematosus.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+strain+differences+in+plasmacytoid+dendritic+cell+frequency+and+function+revealed+by+a+novel+monoclonal+antibody.">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><li>Liao, R., 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=Tacrolimus+Protects+Podocytes+from+Injury+in+Lupus+Nephritis+Partly+by+Stabilizing+the+Cytoskeleton+and+Inhibiting+Podocyte+Apoptosis.">Tacrolimus Protects Podocytes from Injury in Lupus Nephritis Partly by Stabilizing the Cytoskeleton and Inhibiting Podocyte Apoptosis.</a> PLoS One. 10 (7), e0132724 (2015).</li></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 (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.

우리는 높은 순도 골수 PDC를 풍부하게하는 것을 목표로하고 IFNα를 생산하는 능력에 대한 PDC의 기능 변화를 연구하는 젊은이와 노인 모두의 MRL / LPR의 루푸스 발생하기 쉬운 마우스에서 다른 세포 유형의 영향없이. 사용 된 첫 번째 정제 전략은 그림 1과 같이 농축 후 만 7.75 % 순도 주도, MCS했다. MCS에 비해 FACS은 96.4 %로 높은 순도의 PDC 농후. 높은 순도를 보장?...

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

우리는 PDC의 기능 연구를위한 루푸스 발생하기 쉬운 마우스의 골수로부터 고순도 형질 수지상 세포 (PDC)를 분리 정렬 형광 활성화 된 세포를 사용하여 프로토콜을보고합니다.

마우스 골수에서 형질 수지상 세포의 정화를위한 형광 활성화 셀 정렬

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

fluorescence-activated-cell-sorting-for-purification-plasmacytoid

Research

JoVE Journal

Immunology and Infection

12.1K 조회수.  Virginia Polytechnic Institute and State University.  우리는 PDC의 기능 연구를위한 루푸스 발생하기 쉬운 마우스의 골수로부터 고순도 형질 수지상 세포 (PDC)를 분리 정렬 형광 활성화 된 세포를 사용하여 프로토콜을보고합니다. 

비디오: Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

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

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known phenotype.  Other bulk methods of purification include panning, complement depletion and magnetic bead separation.  However, FACS has several advantages over other available methods.  FACS is the preferred method when very high purity of the desired population is required, when the target cell population expresses a very low level of the identifying marker or when cell populations require separation based on differential marker density.  In addition, FACS is the only available purification technique to isolate cells based on internal staining or intracellular protein expression, such as a genetically modified fluorescent protein marker.  FACS allows the purification of individual cells based on size, granularity and fluorescence.  In order to purify cells of interest, they are first stained with fluorescently-tagged monoclonal antibodies (mAb), which recognize specific surface markers on the desired cell population (1).  Negative selection of unstained cells is also possible.  FACS purification requires a flow cytometer with sorting capacity and the appropriate software.  For FACS, cells in suspension are passed as a stream in droplets with each containing a single cell in front of a laser.  The fluorescence detection system detects cells of interest based on predetermined fluorescent parameters of the cells.  The instrument applies a charge to the droplet containing a cell of interest and an electrostatic deflection system facilitates collection of the charged droplets into appropriate collection tubes (2).  The success of staining and thereby sorting depends largely on the selection of the identifying markers and the choice of mAb.  Sorting parameters can be adjusted depending on the requirement of purity and yield.  Although FACS requires specialized equipment and personnel training, it is the method of choice for isolation of highly purified cell populations.

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting FACS

For many scientific studies requiring a biological and chemical analysis of cell populations the cells must be in a high state of purity. Fluorescence activated cell sorting (FACS) is a superior method in which to obtain pure cell populations.

형광 활성 세포 정렬 (외과)에 의해 특정 세포 인구의 정제

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

연구

면역학 및 감염병학

Isolation of Mouse Lung Dendritic Cells

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the respiratory tract. At least three main subsets of lung dendritic cells have been described in mice: conventional DC (cDC) 4, plasmacytoid DC (pDC) 5 and the IFN-producing killer DC (IKDC) 6,7. The cDC subset is the most prominent DC subset in the lung 8. 
The common marker known to identify DC subsets is CD11c, a type I transmembrane integrin (&beta;2) that is also expressed on monocytes, macrophages, neutrophils and some B cells 9. In some tissues, using CD11c as a marker to identify mouse DC is valid, as in spleen, where most CD11c+ cells represent the cDC subset which expresses high levels of the major histocompatibility complex class II (MHC-II). However, the lung is a more heterogeneous tissue where beside DC subsets, there is a high percentage of a distinct cell population that expresses high levels of CD11c bout low levels of MHC-II. Based on its characterization and mostly on its expression of F4&#47;80, an splenic macrophage marker, the CD11chiMHC-IIlo lung cell population has been identified as pulmonary macrophages 10 and more recently, as a potential DC precursor 11. 
In contrast to mouse pDC, the study of the specific role of cDC in the pulmonary immune response has been limited due to the lack of a specific marker that could help in the isolation of these cells. Therefore, in this work, we describe a procedure to isolate highly purified mouse lung cDC. The isolation of pulmonary DC subsets represents a very useful tool to gain insights into the function of these cells in response to respiratory pathogens as well as environmental factors that can trigger the host immune response in the lung.

A highly purified preparation of mouse lung dendritic cells is described. Specific emphasis is given to the isolation of conventional dendritic cell subset.

마우스 폐에 돌기 세포의 분리

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the ...

Preparation of Myeloid Derived Suppressor Cells (MDSC) from Naive and Pancreatic Tumor-bearing Mice using Flow Cytometry and Automated Magnetic Activated Cell Sorting (AutoMACS)

MDSC are a heterogeneous population of immature macrophages, dendritic cells and granulocytes that accumulate in lymphoid organs in pathological conditions including parasitic infection, inflammation, traumatic stress, graft-versus-host disease, diabetes and cancer1-7. In mice, MDSC express Mac-1 (CD11b) and Gr-1 (Ly6G and Ly6C) surface antigens7. It is important to note that MDSC are well studied in various tumor-bearing hosts where they are significantly expanded and suppress anti-tumor immune responses compared to na&iuml;ve counterparts7-10. However, depending on the pathological condition, there are different subpopulations of MDSC with distinct mechanisms and targets of suppression11,12. Therefore, effective methods to isolate viable MDSC populations are important in elucidating their different molecular mechanisms of suppression in vitro and in vivo.
Recently, the Ghansah group has reported the expansion of MDSC in a murine pancreatic cancer model. Our tumor-bearing MDSC display a loss of homeostasis and increased suppressive function compared to na&iuml;ve MDSC 13. MDSC percentages are significantly less in lymphoid compartments of na&iuml;ve vs. tumor-bearing mice. This is a major caveat, which often hinders accurate comparative analyses of these MDSC. Therefore, enriching Gr-1+ leukocytes from na&iuml;ve mice prior to Fluorescence Activated Cell Sorting (FACS) enhances purity, viability and significantly reduces sort time. However, enrichment of Gr-1+ leukocytes from tumor-bearing mice is optional as these are in abundance for quick FACS sorting. Therefore, in this protocol, we describe a highly efficient method of immunophenotyping MDSC and enriching Gr-1+ leukocytes from spleens of na&iuml;ve mice for sorting MDSC in a timely manner. Immunocompetent C57BL/6 mice are inoculated with murine Panc02 cells subcutaneously whereas na&iuml;ve mice receive 1XPBS. Approximately 30 days post inoculation; spleens are harvested and processed into single-cell suspensions using a cell dissociation sieve. Splenocytes are then Red Blood Cell (RBC) lysed and an aliquot of these leukocytes are stained using fluorochrome-conjugated antibodies against Mac-1 and Gr-1 to immunophenotype MDSC percentages using Flow Cytometry. In a parallel experiment, whole leukocytes from na&iuml;ve mice are stained with fluorescent-conjugated Gr-1 antibodies, incubated with PE-MicroBeads and positively selected using an automated Magnetic Activated Cell Sorting (autoMACS) Pro Separator. Next, an aliquot of Gr-1+ leukocytes are stained with Mac-1 antibodies to identify the increase in MDSC percentages using Flow Cytometry. Now, these Gr1+ enriched leukocytes are ready for FACS sorting of MDSC to be used in comparative analyses (na&iuml;ve vs. tumor- bearing) in in vivo and in vitro assays.

Preparation of Myeloid Derived Suppressor Cells MDSC from Naive and Pancreatic Tumor-bearing Mice using Flow Cytometry and Automated Magnetic Activated Cell Sorting AutoMACS

This is a rapid and comprehensive method of immunophenotyping Myeloid Derived Suppressor Cells (MDSC) and enriching Gr-1+ leukocytes from mouse spleens. This method uses flow cytometry and AutoMACS Cell Sorting to enrich for viable Gr-1+ leukocytes prior to FACS sorting of MDSC for use in vivo and in vitro assays.

유동세포계측법 및 자동화된 마그네틱 활성화 셀 정렬 (AutoMACS)를 사용 나이브 및 췌장 종양 베어링 마우스에서 골수양 파생 억제 세포 (MDSC)의 작성

MDSC are a heterogeneous population of immature macrophages, dendritic cells and granulocytes that accumulate in lymphoid organs in pathological ...

의학

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

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and food antigens while concomitantly remaining poised to mount inflammatory responses toward invasive pathogens1,2. Antigen presenting cells, particularly DCs and macrophages, play critical roles in maintaining intestinal immune homeostasis via their ability to sense and appropriately respond to the microbiota3-14. Efficient isolation of intestinal DCs and macrophages is a critical step in characterizing the phenotype and function of these cells. While many effective methods of isolating intestinal immune cells, including DCs and macrophages, have been described6,10,15-24, many rely upon long digestions times that may negatively influence cell surface antigen expression, cell viability, and/or cell yield. Here, we detail a methodology for the rapid isolation of large numbers of viable, intestinal DCs and macrophages. Phenotypic characterization of intestinal DCs and macrophages is carried out by directly staining isolated intestinal cells with specific fluorescence-labeled monoclonal antibodies for multi-color flow cytometric analysis. Furthermore, highly pure DC and macrophage populations are isolated for functional studies utilizing CD11c and CD11b magnetic-activated cell sorting beads followed by cell sorting.

Here, we detail a methodology for the rapid isolation of mouse intestinal dendritic cells (DCs) and macrophages. Phenotypic characterization of intestinal DCs and macrophages is performed using multi-color flow cytometric analysis while magnetic bead enrichment followed by cell sorting is used to yield highly pure populations for functional studies.

마우스 소장에서 돌기 세포 및 Macrophages의 분리 및 특성화

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

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

인간의 흉선에서 골수 수지상 세포와 상피 세포의 분리

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

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

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal Peyer&#39;s patch (PP). Common dendritic cell progenitors (CDPs, lin- c-kitlo CD115+ Flt3+) were purified from the bone marrow of C57BL6 mice by FACS and transferred to recipient mice that lack a significant pDC population in PP; in this case, Ifnar-/- mice were used as the transfer recipients. In some mice, overexpression of the dendritic cell growth factor Flt3 ligand (Flt3L) was enforced prior to adoptive transfer of CDPs, using hydrodynamic gene transfer (HGT) of Flt3L-encoding plasmid. Flt3L overexpression expands DC populations originating from transferred (or endogenous) hematopoietic progenitors. At 7-10 days after progenitor transfer, pDCs that arise from the adoptively transferred progenitors were distinguished from recipient cells on the basis of CD45 marker expression, with pDCs from transferred CDPs being CD45.1+ and recipients being CD45.2+. The ability of transferred CDPs to contribute to the pDC population in PP and to respond to Flt3L was evaluated by flow cytometry of PP single cell suspensions from recipient mice. This method may be used to test whether other progenitor populations are capable of generating PP pDCs. In addition, this approach could be used to examine the role of factors that are predicted to affect pDC development in PP, by transferring progenitor subsets with an appropriate knockdown, knockout or overexpression of the putative developmental factor and/or by manipulating circulating cytokines via HGT. This method may also allow analysis of how PP pDCs affect the frequency or function of other immune subsets in PPs. A unique feature of this method is the use of Ifnar-/- mice, which show severely depleted PP pDCs relative to wild type animals, thus allowing reconstitution of PP pDCs in the absence of confounding effects from lethal irradiation.

This protocol describes experimental procedures to assess the differentiation of plasmacytoid dendritic cells in Peyer&#8217;s patch from common dendritic cell progenitors, using techniques involving FACS-mediated cell isolation, hydrodynamic gene transfer, and flow analysis of immune subsets in Peyer&#8217;s patch.

조혈 전구 세포의 입양 전송을 사용 Peyer의 패치에 쥐과 Plasmacytoid 수지상 세포의 개발을 평가

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

Plasmacytoid dendritic cells (pDCs) are powerful type I interferon (IFN-I) producing cells that are activated in response to infection or during inflammatory responses. Unfortunately, study of pDC function is hindered by their low frequency in lymphoid organs, and existing methods for in vitro DC generation predominantly favor the production of cDCs over pDCs. Here we present a unique approach to efficiently generate pDCs from common lymphoid progenitors (CLPs) in vitro. Specifically, the protocol described details how to purify CLPs from bone marrow and generate pDCs by coculturing with &#947;-irradiated AC-6 feeder cells in the presence of Flt3 ligand. A unique characteristic of this culture system is that the CLPs migrate underneath the AC-6 cells and become cobblestone area-forming cells, a critical step for expanding pDCs. Morphologically distinct DCs, namely pDCs and cDCs, were generated after approximately 2 weeks with a composition of 70-90% pDCs under optimal conditions. Typically, the number of pDCs generated by this method is roughly 100-fold of the number of CLPs seeded. Therefore, this is a novel system with which to robustly generate the large numbers of pDCs required to facilitate further studies into the development and function of these cells.

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

In this study we present an in vitro culture system that can efficiently generate pDCs by co-culturing common lymphoid progenitors with AC-6 feeder cells in the presence of Flt3 ligand.

AC-6 공급 시스템을 사용하여 일반 림프 전구 세포에서 쥐 형질 수지상 세포의 체외 세대

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

An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen presenting molecules to initiate T-cell-mediated immunity. Dendritic cells can be separated into several phenotypically and functionally heterogeneous subsets. Three important subsets of splenic dendritic cells are plasmacytoid, CD8&#945;Pos and CD8&#945;Neg cells. The plasmacytoid DCs are natural producers of type I interferon and are important for anti-viral T cell immunity. The CD8&#945;Neg DC subset is specialized for MHC class II antigen presentation and is centrally involved in priming CD4 T cells. The CD8&#945;Pos DCs are primarily responsible for cross-presentation of exogenous antigens and CD8 T cell priming. The CD8&#945;Pos DCs have been demonstrated to be most efficient at the presentation of glycolipid antigens by CD1d molecules to a specialized T cell population known as invariant natural killer T (iNKT) cells. Administration of Flt-3 ligand increases the frequency of migration of dendritic cell progenitors from bone marrow, ultimately resulting in expansion of dendritic cells in peripheral lymphoid organs in murine models. We have adapted this model to purify large numbers of functional dendritic cells for use in cell transfer experiments to compare in vivo proficiency of different DC subsets.

Distinct dendritic cell subsets exist as rare populations in lymphoid organs, and therefore are challenging to isolate in sufficient numbers and purity for immunological experiments. Here we describe a high efficiency, high yield method for isolation of all of the currently known major subsets of mouse splenic dendritic cells.

마우스 수지상 세포 하위 집합의 분리를위한 효율적이고 높은 수율 방법

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen ...

Economical and Efficient Protocol for Isolating and Culturing Bone Marrow-derived Dendritic Cells from Mice

The demand for dendritic cells (DCs) is gradually increasing as immunology research advances. However, DCs are rare in all tissues. The traditional method for isolating DCs primarily involves inducing bone marrow (BM) differentiation into DCs by injecting large doses (&#62;10 ng/mL) of granulocyte-macrophage colony-stimulating factor/interleukin-4 (GM-CSF/IL-4), making the procedure complex and expensive. In this protocol, using all BM cells cultured in 10 ng/mL GM-CSF/IL-4 medium, after 3-4 half-culture exchanges, up to 2.7 x 107 CD11c+ cells&#160;(DCs) per mouse (two femurs) were harvested with a purity of 80%-95%. After 10 days in culture, the expression of CD11c, CD80, and MHC II increased, whereas the number of cells decreased. The number of cells peaked after 7 days of culture. Moreover, this method only took 10 min to harvest all bone marrow cells, and a high number of DCs were obtained after 1 week of culture.

Here, we present an economical and efficient method to isolate and generate high-purity bone marrow-derived dendritic cells from mice after 7 days of culture with 10 ng/mL GM-CSF/IL-4.

마우스로부터 골수 유래 수지상 세포를 분리하고 배양하기 위한 경제적이고 효율적인 프로토콜

The demand for dendritic cells (DCs) is gradually increasing as immunology research advances. However, DCs are rare in all tissues. The traditional method ...

In Vitro Generation of Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors

Source: Chang, S., et al. <a href="https://app.jove.com/v/53211">In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System.</a> J. Vis. Exp. (2015)
This video demonstrates an in vitro assay to generate plasmacytoid dendritic cells (pDCs) from common lymphoid progenitor cells (CLPs). Upon co-culturing purified CLPs with &#947;-irradiated feeder cells in the presence of Fms-like tyrosine kinase-3 ligand (FL), the CLPs differentiate into non-adherent pDCs, the presence of which is confirmed using flow cytometry.

In vitro Generation of Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors(일반 림프구 전구 세포에서 Plasmacytoid 수지상 세포의 체외 생성)

Source: Chang, S., et al. In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System. J. ...