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>

<ol>
	<li>Bonner, W. A., Hulett, H. R., Sweet, R. G., Herzenberg, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+activated+cell+sorting.">Fluorescence activated cell sorting.</a> Rev Sci Instrum. 43 (3), 404-409 (1972).</li><li>Herzenberg, L. 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=The+history+and+future+of+the+fluorescence+activated+cell+sorter+and+flow+cytometry:+a+view+from+Stanford.">The history and future of the fluorescence activated cell sorter and flow cytometry: a view from Stanford.</a> Clin Chem. 48 (10), 1819-1827 (2002).</li><li>Brown, M., Wittwer, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry:+principles+and+clinical+applications+in+hematology.">Flow cytometry: principles and clinical applications in hematology.</a> Clin Chem. 46 (8 Pt 2), 1221-1229 (2000).</li><li>Laerum, O. D., Farsund, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+application+of+flow+cytometry:+a+review.">Clinical application of flow cytometry: a review.</a> Cytometry. 2 (1), 1-13 (1981).</li><li>Alvarez-Barrientos, A., Arroyo, J., Canton, R., Nombela, C., Sanchez-Perez, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+flow+cytometry+to+clinical+microbiology.">Applications of flow cytometry to clinical microbiology.</a> Clin Microbiol Rev. 13 (2), 167-195 (2000).</li><li>Mattanovich, D., Borth, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+cell+sorting+in+biotechnology.">Applications of cell sorting in biotechnology.</a> Microb Cell Fact. 5 (12), (2006).</li><li>Aldahlawi, A. M., Elshal, M. F., Damiaiti, L. A., Damanhori, L. H., Bahlas, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+CD95+and+CCR7+expression+on+circulating+CD4(+)+lymphocytes+revealed+disparate+immunoregulatory+potentials+in+systemic+lupus+erythematosus.">Analysis of CD95 and CCR7 expression on circulating CD4(+) lymphocytes revealed disparate immunoregulatory potentials in systemic lupus erythematosus.</a> Saudi J Biol Sci. 23 (1), 101-107 (2016).</li><li>Cullender, T. C., 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=Innate+and+adaptive+immunity+interact+to+quench+microbiome+flagellar+motility+in+the+gut.">Innate and adaptive immunity interact to quench microbiome flagellar motility in the gut.</a> Cell Host Microbe. 14 (5), 571-581 (2013).</li><li>Fernandez, A. 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

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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.

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

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

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone marrow, spleen, lymph nodes and Peyer's patches 1-3. DCs present in peripheral tissues sample the organism for the presence of antigens, which they take up, process and present in their surface in the context of major histocompatibility molecules (MHC). Then, antigen-loaded DCs migrate to immunological organs where they present the processed antigen to T lymphocytes triggering specific immune responses. One way to evaluate the migratory capabilities of DCs is to label them with fluorescent dyes 4.
Herewith we demonstrate the use of Qdot fluorescent nanocrystals to label murine bone marrow-derived DC. The advantage of this labeling is that Qdot nanocrystals possess stable and long lasting fluorescence that make them ideal for detecting labeled cells in recovered tissues. To accomplish this, first cells will be recovered from murine bone marrows and cultured for 8 days in the presence of granulocyte macrophage-colony stimulating factor in order to induce DC differentiation. These cells will be then labeled with fluorescent Qdots by short in vitro incubation. Stained cells can be visualized with a fluorescent microscopy. Cells can be injected into experimental animals at this point or can be into mature cells upon in vitro incubation with inflammatory stimuli. In our hands, DC maturation did not determine loss of fluorescent signal nor does Qdot staining affect the biological properties of DCs. Upon injection, these cells can be identified in immune organs by fluorescent microscopy following typical dissection and fixation procedures.

Dendritic cells uptake antigens and migrate towards immune organs to present processed antigens to T cells. Qdot nanocrystal labeling provides a long-lasting and stable fluorescent signal. This allows tracking of dendritic cells to different organs by fluorescent microscopy.

추적 연구 Qdot Nanocrystals와 Murine 골수 파생 돌기 세포의 생성 및 라벨링

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

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

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 to colitis. If the gene-of-interest is expressed in both bone marrow derived cells and non-bone marrow derived cells of the host; however, it is possible to differentiate the role of a gene of interest in bone marrow derived cells and non- bone marrow derived cells by bone marrow transplantation technique. To change the bone marrow derived cell genotype of mice, the original bone marrow of recipient mice were destroyed by irradiation and then replaced by new donor bone marrow of different genotype. When wild-type mice donor bone marrow was transplanted to knockout mice, we could generate knockout mice with wild-type gene expression in bone marrow derived cells. Alternatively, when knockout mice donor bone marrow was transplanted to wild-type recipient mice, wild-type mice without gene-of-interest expressing from bone marrow derived cells were produced. However, bone marrow transplantation may not be 100% complete. Therefore, we utilized cluster of differentiation (CD) molecules (CD45.1 and CD45.2) as markers of donor and recipient cells to track the proportion of donor bone marrow derived cells in recipient mice and success of bone marrow transplantation. Wild-type mice with CD45.1 genotype and knockout mice with CD45.2 genotype were used. After irradiation of recipient mice, the donor bone marrow cells of different genotypes were infused into the recipient mice. When the new bone marrow regenerated to take over its immunity, the mice were challenged by chemical agent (dextran sodium sulfate, DSS 5%) to induce colitis. Here we also showed the method to induce colitis in mice and evaluate the role of the gene of interest expressed from bone-marrow derived cells. If the gene-of-interest from the bone derived cells plays an important role in the development of the disease (such as colitis), the phenotype of the recipient mice with bone marrow transplantation can be significantly altered. At the end of colitis experiments, the bone marrow derived cells in blood and bone marrow were labeled with antibodies against CD45.1 and CD45.2 and their quantitative ratio of existence could be used to evaluate the success of bone marrow transplantation by flow cytometry. Successful bone marrow transplantation should show a vast majority of donor genotype (in term of CD molecule marker) over recipient genotype in both the bone marrow and blood of recipient mice.

Bone marrow transplantation provides a way to change the genotype of the bone marrow derived cells. If the gene of interest is expressed in both bone marrow derived cells and non-bone marrow derived cells, bone marrow transplantation can change the bone marrow derived cells to a different genotype without changing the non-bone marrow derived cell genotype.

골수 이식으로 마우스 대장염에서 면역 및 비 면역 세포에서 유전자 발현의 기능 역할을 차별화

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

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or pathogenic effects depending on the inflammatory milieu. Nonetheless, despite the indispensable role of neutrophils in immunity, detailed understanding of the molecular factors that mediate neutrophils' effector and immunopathogenic effects in different infectious diseases and inflammatory conditions is still lacking, partly because of their short half life, the difficulties with handling of these cells and the lack of reliable experimental protocols for obtaining sufficient numbers of neutrophils for downstream functional studies and adoptive transfer experiments. Therefore, simple, fast, economical and reliable methods are highly desirable for harvesting sufficient numbers of mouse neutrophils for assessing functions such as phagocytosis, killing, cytokine production, degranulation and trafficking. To that end, we present a reproducible density gradient centrifugation-based protocol, which can be adapted in any laboratory to isolate large numbers of neutrophils from the bone marrow of mice with high purity and viability. Moreover, we present a simple protocol that uses CellTracker dyes to label the isolated neutrophils, which can then be adoptively transferred into recipient mice and tracked in several tissues for at least 4 hr post-transfer using flow cytometry. Using this approach, differential labeling of neutrophils from wild-type and gene-deficient mice with different CellTracker dyes can be successfully employed to perform competitive repopulation studies for evaluating the direct role of specific genes in trafficking of neutrophils from the blood into target tissues in vivo.

We describe a protocol for isolation and purification of neutrophils from mouse bone marrow by density gradient centrifugation and for neutrophil labeling using CellTracker dyes. This represents a simple, fast, reproducible and economical method for obtaining large numbers of neutrophils for downstream functional studies or adoptive transfer and tracking experiments.

분리, 정제 및 기능 연구 및 입양 전송 실험 쥐의 골수 호중구의 라벨링

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

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 ...

Tracking Mouse Bone Marrow Monocytes In Vivo

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal environment. Biological activities of immune cells mainly rely on their motility capacities. Blood monocytes have short half-life in the bloodstream; they originate in the bone marrow and are constitutively released from it. In inflammatory condition, this process is enhanced, leading to blood monocytosis and subsequent infiltration of the peripheral inflammatory tissues. Identifying the biomechanical events controlling monocyte trafficking from the bone marrow towards the vascular network is an important step to understand monocyte physiopathological relevance. We performed in vivo time-lapse imaging by two-photon microscopy of the skull bone marrow of the Csf1r-Gal4VP16/UAS-ECFP (MacBlue) mouse. The MacBlue mouse expresses the fluorescent reporters enhanced cyan fluorescent protein (ECFP) under the control of a myeloid specific promoter 1, in combination with vascular network labelling. We describe how this approach enables the tracking of individual medullar monocytes in real time to further quantify the migratory behaviour within the bone marrow parenchyma and the vasculature, as well as cell-to-cell interactions. This approach provides novel insights into the biology of the bone marrow monocyte subsets and allows to further address how these cells can be influenced in specific pathological conditions.

Tracking Mouse Bone Marrow Monocytes In Vivo

Monocytes are key regulators of innate immunity and play a critical role in the renewal of the peripheral mononuclear phagocytic system and in case of inflammation. This manuscript describes the procedure of real time imaging of the mouse calvaria bone marrow to study the monocyte mobilisation mechanism.

마우스 골수 단핵구를 추적<em&gt; 생체</em

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal ...

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 ...

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

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. However, they are difficult to isolate in sufficient numbers to study them in detail, therefore, in vitro models have been developed. In vitro cultures of bone marrow-derived macrophages and dendritic cells are well-established and valuable methods for immunological studies. Here, a method for culturing and identifying both DCs and macrophages from a single culture of primary mouse bone marrow cells using the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF) is described. This protocol is based on the established procedure first developed by Lutz et al. in 1999 for bone marrow-derived DCs. The culture is heterogeneous, and MHCII and fluoresceinated hyaluronan (FL-HA) are used to distinguish macrophages from immature and mature DCs. These GM-CSF derived macrophages provide a convenient source of in vitro derived macrophages that closely resemble alveolar macrophages in both phenotype and function.

Bone marrow cells cultured with granulocyte macrophage colony stimulating factor (GM-CSF) generate a heterogeneous culture containing macrophages and dendritic cells (DCs). This method highlights using MHCII and hyaluronan (HA) binding to differentiate macrophages from the DCs in the GM-CSF culture. Macrophages in this culture have many similarities to alveolar macrophages.

마우스 골수에서 생성 및 GM-CSF의 식별 파생 폐포와 같은 대 식세포와 수지상 세포

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

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have recently been recognized to be more heterogeneous than previously appreciated. These cultures routinely contain moDC as well monocyte-derived macrophages (moMac), and even some less developed cells such as monocytes. The goal of this protocol is to provide a consistent method for identification and separation of the many cell types present in these cultures as they develop, so that their specific functions may be further investigated. The sorting strategy presented here separates cells first into four populations based on expression of Ly6C and CD115, both of which are expressed transiently by cells as they develop in GM-CSF-driven culture. These four populations include Common myeloid progenitors or CMP (Ly6C-, CD115-), granulocyte/macrophage progenitors or GMP (Ly6C+, CD115-), monocytes (Ly6C+, CD115+), and monocyte-derived macrophages or moMac (Ly6C-, CD115+). CD11c is also added to the sorting strategy to distinguish two populations within the Ly6C-, CD115- population: CMP (CD11c-) and moDC (CD11c+). Finally, two populations may be further distinguished within the Ly6C-, CD115+ population based on the level of MHC class II expression. MoMacs express lower levels of MHC class II, while a monocyte-derived DC precursor (moDP) expresses higher MHC class II. This method allows for the reliable isolation of several developmentally distinct populations in numbers sufficient for a variety of functional and developmental analyses. We highlight one such functional readout, the differential responses of these cell types to stimulation with Pathogen-Associated Molecular Patterns (PAMPs).

Here we provide a method for identifying and isolating large numbers of GM-CSF driven myeloid cells using high speed cell sorting. Five distinct populations (Common myeloid progenitors, granulocyte/macrophage progenitors, monocytes, monocyte-derived macrophages, and monocyte-derived DCs) can be identified based on Ly6C and CD115 expression.

골수성 창시자 및 정렬 전략 소설 셀을 사용 하 여 Murine 골에서 수지상 세포 선구자의 큰 숫자의 생성

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

An In Vivo Mouse Model to Measure Na&#239;ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played by T cells in an immune response. This protocol describes the in vitro differentiation of bone marrow (BM) progenitors to obtain granulocyte macrophage colony-stimulating factor (GM-CSF) derived-dendritic cells (DCs). The protocol also describes the adoptive transfer of ovalbumin peptide (OVAp)-loaded GM-CSF-derived DCs and na&#239;ve CD4 T cells from OTII transgenic mice in order to analyze the in vivo activation, proliferation, and Th1 differentiation of the transferred CD4 T cells. This protocol circumvents the limitation of purely in vivo methods imposed by the inability to specifically manipulate or select the studied cell population. Moreover, this protocol allows studies in an in vivo environment, thus avoiding alterations to functional factors that may occur in vitro and including the influence of cell types and other factors only found in intact organs. The protocol is a useful tool for generating changes in DCs and T cells that modify adaptive immune responses, potentially providing important results to understand the origin or development of numerous immune associated diseases.

Here, we present a protocol for the in vivo determination of na&#239;ve CD4 T cell (T cell) activation, proliferation, and Th1 differentiation induced by GM-CSF bone marrow (BM)-derived dendritic cells (DCs). In addition, this protocol describes BM and T-cell isolation, DC generation, and DC and T-cell adoptive transfer.

Vivo에서 마우스 모델을 측정 순진한 CD4 T 세포 활성화, 확산 및 골 수 유래 수지상 세포에 의해 유도 된 Th1 차별화

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played ...