우리는 마우스 처리와 관련하여 도움이 ultracentrifugation와 패티 키서와 관련하여 도움이 스티브 McBryant와 제프 핸슨 감사드립니다. 국립 보건원의 신경 질환 및 뇌졸중의 국립 연구소, 부여 5R01NS056379 - 02이 작품을 후원.

<html> 1. 프리온 봉 정화 및 라벨링 이 프로토콜은 한 이전에 출판 18 적응됩니다 <ol><li> 상업 믹서의 최대 속도로 1 분 후, 얼음에 2 분 얼음 추위 homogenizing 버퍼의 900 ML에서 프리온에 감염된 뇌 조직 100g (HB, 1X PBS 320 MM의 자당, 150 MM NaCl 및 4mm짜리 EDTA (에틸렌 다이아 민 테트라 초산)을 포함하는)를 균질. 세 번 반복합니다. </li><li> 10 3,000 XG에서 분 및 4 ° C.에 대한 homogenate 원심 분리기 제거하고 얼음에 뜨는 저장합니다. 1L HB의 펠렛을 다시 정지 및 단계 1.1과 1.2를 반복합니다. </li><li> 100,000 XG, 0 ° C. 60 분 풀 supernatants 및 원심 분리기 뜨는 폐기하십시오. </li><li> 다시 중지 1X 트리스의 100 ML의 펠렛은 (10 MM 트리스 - HCL, 0.1 MM NaCl 1 MM EDTA (에틸렌 다이아 민 테트라 초산))이 2퍼센트 트리톤 X - 100 (TBST)이있는 염분 버퍼. 견적 단백질 브래드 또는 이와 유사한 분석하여 농도 5 TBST에 MG / ML을 조절할 수 있습니다. 30 분 얼음 칠. </li><li> 100,000 XG, 0 ° C.에서 30 분 원심 분리기 뜨는 취소 50 ML의 TBST 다음 100 ML TBS와 펠렛을 씻으십시오. 100 ML 1X PBS 1 %의 sarcosyl 및 테아제 억제제 칵테일을 포함에서 펠렛을 다시 정지 37에서 120 분 저어 ° C. </li><li> 100,000 XG, 10 ° C. 60 분 320 MM의 자당과 원심 분리기 900 ML에 레이어 샘플 뜨는 취소 10 ML 2.3M NaCl에 다시 중지 펠렛, 5 % sarcosyl. </li><li> 얼음 1 분 간격으로 70 % 최대 전력에서 샘플 5 X 40 초 Sonicate. </li><li> 13,000 XG, 4oC에서 15 분 동안 eppendorf 튜브 및 원심 분리기에 샘플 나누어지는 1 ML. TBS 및 매장과 함께 두 번 알약을 씻어 @ -70 ° C 또는 단계 1.9에서 fluorochrome에 공액. </li><li> DyLight 649 1 병을 사용하여 프리온 봉 정화의 공액 한 튜브 제조 업체의 프로토콜에 따라 fluorochrome. </li><li> 필터 컬럼을 통해 투석이나 원심 분리하여 PBS에 대한 Exchange DyLight 라벨 버퍼. -70 ° C에서 멀리 사용할까지 빛으로부터 20 μL aliquots에 보관하십시오. </li></ol> 2. 프리온 접종 및 셀 복구 이 섹션은 intraperitoneal 프리온 접종과 복막, mediastinal 및 mesenteric 림프절과 비장에서 세포의 복구를 포함. <ol><li> 사용하는 즉시 이전 형광 프리온 막대 PBS에서 1시 50분을 희석. 엄지와 검지로 지느러미 목 모피로 아저씨의 마우스는 부드럽게 얼굴과 분홍색 손가락으로 꼬리를 묶어 두려고 마우스를 켜십시오. 머리 위에서 뒷다리 몸통 이팅 및 약간 거꾸로 그를 잡고 마우스를 Supinate. </li><li> 30G의 인슐린 주사기를 사용하여 sacroiliac 관절에 앞쪽에 정중선과 1~2cm의 1cm 측면 형광 프리온 막대 100 μL를 주입. 피부를 통해서도 1cm가 medially 45 °를 감독 바늘을 삽입합니다. 컨트롤로 형광 구슬 또는 PBS 혼자의 1시 50분 희석을 사용합니다. 분석하기 전에 할당된 시간 쥐를 품어. </li><li> 승인 동물 보호에 따라 마우스를 안락사 및위원회 프로토콜을 사용합니다. 12 ML 주사기와 25G 바늘을 사용하여 복막 구멍에 PBS 10 ML을 주사. 부드럽게 일분 길이 방향 시체를 바위. 지육 절개가 지속하는 동안 얼음에 15 ML 원뿔 관과 보유에 동일한 주사기와 16G 바늘을 사용하는 복막의 세포 현탁액을 복구합니다. </li><li> 70 % 에탄올로 철저하게 시체의 복부 측면 젖었어. 그냥 집게로 ribcage 아래의 정중선에서 피부를 허용하고 가위로 피부를 통해 작은 절개를 잘라. 각 엔드에서 정중선에서 두 2 cm의 incisions 옆으로 후, 머리와 꼬리 양쪽 방향에서 피부를 통해 3~5cm 긴 절개를, 거기서부터. 다시 필과 복막 및​​ 흉부 충치를 나타내기 위해 피부를 핀. 조심스럽게 복막을 둘러싸고있는 얇은 반투명 세포막을 통해 잘라 폐종격부을 나타내기 위해 흉부를 반영합니다. </li><li> 기관의 복부 쪽 근처 brachiocephalic 동맥 medially 인접한 thymus에 약간 지느러미와 옆으로 인접 2-4 mediastinal 림프절을,,, 찾기 및 제거합니다. 찾기 및 부드러운 흰색 mesenteric 조직에 포함된 여섯 mesenteric의 림프절까지 제거하는 사후 복부 벽에 앵커는 내장. 비장, 단지 정중선의 측면과 복부의 왼쪽에있는 내장에있는 등 지느러미 긴 어두운 빨간색 기관을 찾아 제거합니다. 예약 림프절 얼음 1 ML RPMI 1640 배지에 절개가 완료될 때까지. </li><li> 더운 물에 담가서 부드럽게하고 1 ML 주사기의 플런저를 사용하여 플라스틱 페트리 접시에 RPMI 3 ML로 40 μm의 메쉬 스트레이너를 통해 림프절을 누르십시오. RPMI의 추가 2 ML와 스트레이너를 씻어 15 ML 원뿔 관에 5 ML 세포 현탁액을 전송할 수 있습니다. RPMI의 추가 5 ML있는 페트리 접시를 씻어 같은 튜브로 전송할 수 있습니다. 250 XG에서 5 분 원심 분리기, 1 ML 외과 버퍼 (1X PBS, 1 % 소 혈청 알부민 및 10 MM EDTA (에틸렌 다이아 민 테트라 초산))에 뜨는을 다시 중단 세포 펠렛을 버리고 1.5 ML Eppendorf 튜브에 전송할 수 있습니다. </li></ol><html> ove_title &quot;&gt; 3. 세포 스테 이닝 및 유동세포계측법 다음은 잘 특징인 면역 세포 표면 마커에 대한 형광 항체를 사용하여 면역 세포 하위 집합을 식별에 대한 일반적인 얼룩 프로토콜입니다. 모든 항체는 사용하기 전에 즉시 외과의 버퍼로 희석되었다. <ol><li> Eppendorf 튜브에 세포 나누어지는 10 6 세포를 계산합니다. 원심 분리기 세포 (2.7 참조​​) 1 ML 외과 버퍼로 세척 펠릿 2X. </li><li> 내생 FC 수용체를 차단 2 NG / ML 쥐 방지 마우스 CD16/32 100 μL의 세포에게 얼음에 20 분 알을 품다. 펠렛 및 외과 버퍼로 세척 세포. </li><li> 한 시간 만이라도 얼음의 각 샘플과 부화 1ng / ML 적절한 형광등 antibod (Y) 이거야 100 μL를 추가합니다. 제어 세포에 혼자 외과 버퍼 100 μL를 추가합니다. 펠렛 및 외과 버퍼로 두 번 세척 세포. </li><li> 1 ML ACK 버퍼 (NH 4 Club 호텔 150 MM, 1 MM KHCO 3 0.1 MM EDTA (에틸렌 다이아 민 테트라 초산)) 적혈구를 lyse 1 분 얼음에 품어에서 다시 중지 세포. 펠렛 및 외과 버퍼로 세척 세포. 1 ML 외과 버퍼의 세포를 다시 일시 중지합니다. </li><li> 일반적으로 405, 488 및 633 나노미터 여기 레이저 5-10 형광 방출 탐지기로, 여러 가지 빛깔의 검출 및 분석을 수있는 흐름 cytometer를 사용하여 세포에서 데이터를 수집합니다. </li></ol> 4. 대표 결과 : 우리는 원유에서 프리온 봉 정화, E2 엘크 뇌 homogenate (차선 1 및 2)의 프리온에 감염된 뇌 homogenate (그림 1) 세제 solubilization 및 ultracentrifugation 크게 또한 프로 테아제 K되었습니다 프리온 막대 (차선 1 3 비교)에 대한 풍부한 저항 (레인 4). 흥미롭게도, 소화되지 않은 정화 프리온 막대는 prions에 대한 극적인 농축을 나타내는 소화 E2 뇌 homogenate에 놀랍도록 닮아 glycoform 프로필을 표시합니다. 동일 - 대우 정상 뇌 homogenate는 PK 방지 PrP C를 (차선 5 6) 생산하지 않습니다. 특정 세포 집단의 흐름 cytometric 분석은 항원 두 시간에 의해 복막에 세포를 집으로 발표하고 형광 구슬과 프리온 막대로 PBS 주입 제어를 통해 극적으로 증가 형광 (그림 2)에 의해 입증을 유지 보여줍니다. 동안 구슬 (Figs. 2 시간 - N)에서 세포 프리온 (Figs. 2O - U) - 주사 상당한 형광 표시 마우스 (Figs. 2A - G) 배경 위에 형광 표시 PBS - 취급 컨트롤의 복막에서 수확 대한 세포도 특히 monocytes에 (Figs. 2I와 P), 돌기 세포 (Figs. 2K 및 R) 및 macrophages (Figs. 2L 및 S), 일부 Neutrophils (그림 2J 및 Q)와 적은 B 세포 (그림 2T). 비드와 프리온 베어링 monocytes, 돌기 세포 및 macrophages도 이시간 접종 (Figs. 2AD 및 AK, AF 및 AM과 AG와, 각각), 동안 거의 또는 전혀 PrP CWD 베어링 Neutrophils 후 mediastinal 림프절에서 발견되었습니다 , B 또는 T 세포 (Figs. 2AE와 AL, AH 및 AG 각각 AI와 AP)가 발견되었습니다. 우리는 비장이나 림프절 mesenteric (데이터가 표시되지 않음)에 구슬이나 프리온 막대를 감지하지 않습니다. 토토, 이러한 데이터는 면역 세포의 트래픽이 다른 미립자 항원 매우 비슷하게 prions 것을 보여줍니다. <table border="1"><tbody><tr><td> 시약 </td><td> FLUOROCHROME </td><td> 여기 레이저 (NM) </td><td> PEAK 방출 (NM) </td></tr><tr><td> 프리온 막대 </td><td> DyLight 649 </td><td> 633 </td><td> 674 </td></tr><tr><td> 1 μm의 구슬 </td><td> AlexaFluor 660 </td><td> 633 </td><td> 685 </td></tr><tr><td> 항체 </td><td></td><td></td><td></td></tr><tr><td> CD11b </td><td> eFluor 450 </td><td> 405 </td><td> 450 </td></tr><tr><td></td><td> Phycoerythrin - Cy7 </td><td> 488 </td><td> 760 </td></tr><tr><td> CD11c </td><td> R - Phycoerythrin </td><td> 488 </td><td> 575 </td></tr><tr><td></td><td> Phycoerythrin - Cy7 </td><td> 488 </td><td> 760 </td></tr><tr><td> Ly6C </td><td> Fluoroisothiocyante </td><td> 488 </td><td> 518 </td></tr><tr><td> Ly6G </td><td> R - Phycoerythrin </td><td> 488 </td><td> 575 </td></tr><tr><td> CD21 </td><td> DyLight 488 </td><td> 488 </td><td> 518 </td></tr><tr><td> B220 </td><td> Allophycocyanin - Cy7 </td><td> 633 </td><td> 785 </td></tr><tr><td> 인 경우에는 3 번 CD에 들어 </td><td> R - Phycoerythrin </td><td> 488 </td><td> 575 </td></tr></tbody></table>형광 시약의 &quot;&gt; 표 1. 스펙트럼 특성 <img src="/files/ftp_upload/2349/2349fig1.jpg" alt="그림 1" /> 그림 1. 감염된 뇌 homogenate에서 프리온 막대의 정제가. 우리는 집계 프리온 봉 (차선 3, 4) 투석을하는 자료를 시작으로 CWD에 감염된 사슴 (E2, 차선 1and 2)에서 10 % 원유의 뇌 homogenate를 사용했습니다. 정상적인 두뇌 homogenate이 (차선 5 6)하지 않는 반면, - 원유 및 정화 재료는 모두 프로 테아제 K 치료 (4 차선 1) 특성 저항을 보여주었다. 분자량 마커는 얼룩의 왼쪽에 KD에 표시됩니다. BH, 뇌 homogenate. <img src="/files/ftp_upload/2349/2349fig2.jpg" alt="그림 2" /> 그림 2. 접종은. (패널 AG와 V - AB), 형광 구슬 (HN 및 AC - AI) 또는 프리온 막대는 (OU와 AJ - AP = 연합 뉴스) PBS되었습니다 두 시간 후에 복막에서 mediastinal 림프절에 면역 세포의 인신 매매 prions의 흐름 cytometric 분석 복막 세척 유체 (AU) 또는 두 시간 후에 mediastinal 림프절 (V - AP)에서 수확 마우스 및 세포의 복막으로 주입. PBS (패널 A와 V), 형광 구슬 (패널 H와 AC)과 프리온 막대 (패널 O 및 AJ) 대우 생쥐에서 첫 번째 칼럼보기 세포의 그래프로 보여줍니다. 형광 셀 (빨간색 또는 녹색 점들)와 전체 세포 (회색 점) 상대 크기 (앞으로 분산형), 입도 (사이드 분산형) 및 형광 총 라이브 세포의 비율을 보여주 꾸몄다 있습니다. granulocytes의 특징 세포의 중요한 숫자는 형광 구슬과 막대를 유지. 세포는 또한 면역 세포 표면 마커와 LyG - Ly6C + monocytes에 대한 문이 (패널 B, I, P, W, 광고 및 AK), Ly6c - CD11c - CD11b + Ly6G + neutrophils (C, J, Q, X, AE에 대한 항체 물들일되었습니다 그리고 AL), Ly6G - Ly6C - CD11b + CD11c + 돌기 세포 (D, K, R, Y, AF와 AM), Ly6G - Ly6C - CD11c - CD11b + macrophages, (E, L, S, Z, AG와) 인 경우에는 3 번 CD에 들어 - B220 + CD21 + B - 세포 (F, M, T, AA, AH 및 AO) 및 CD21 - B220 - 인 경우에는 3 번 CD + T 세포 (G, N, U, AB, AI 및 AP). Monocytes, 돌기 세포 및 macrophages은 복막 캐비티 (패널 I와 P, K와 R과 L과 S, 각각)에서 형광 구슬과 프리온 봉을 보유하고 mediastinal 림프절 (패널 광고 및 AK, AF 및 오전 및 AG에게 운반 각각),이 두 입자의 유사한 이해와 인신 매매를 시연. Neutrophils은 구슬 (J)와 적은 봉 (Q)의 상당한 금액을 유지하지만, (AE 및 AL) 림프절에게 전달하지 못했습니다. B 세포 일부 봉 (T 및 AO)하지만 거의없이 구슬을 (M 및 AH) 유지 및 수송. T 세포는 구슬이나 막대 (N, U, AI 및 AP = 연합 뉴스)도없는 인신 매매.

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	<li>Klein, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+crucial+role+for+B+cells+in+neuroinvasive+scrapie.">A crucial role for B cells in neuroinvasive scrapie.</a> Nature. 390, 687-687 (1997).</li><li>Mabbott, N. A., Farquhar, C. F., Brown, K. L., Bruce, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+the+immune+system+in+TSE+pathogenesis.">Involvement of the immune system in TSE pathogenesis.</a> Immunol Today. 19, 201-201 (1998).</li><li>Sigurdson, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PrP(CWD)+lymphoid+cell+targets+in+early+and+advanced+chronic+wasting+disease+of+mule+deer.">PrP(CWD) lymphoid cell targets in early and advanced chronic wasting disease of mule deer.</a> J Gen Virol. 83, 2617-2617 (2002).</li><li>Klein, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complement+facilitates+early+prion+pathogenesis.">Complement facilitates early prion pathogenesis.</a> Nat Med. 7, 488-488 (2001).</li><li>Mabbott, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporary+depletion+of+complement+component+C3+or+genetic+deficiency+of+C1q+significantly+delays+onset+of+scrapie.">Temporary depletion of complement component C3 or genetic deficiency of C1q significantly delays onset of scrapie.</a> Nat Med. 7, 485-485 (2001).</li><li>Zabel, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromal+Complement+Receptor+CD21/35+Facilitates+Lymphoid+Prion+Colonization+and+Pathogenesis.">Stromal Complement Receptor CD21/35 Facilitates Lymphoid Prion Colonization and Pathogenesis.</a> J Immunol. 179, 6144-6144 (2007).</li><li>Cordier-Dirikoc, S., Chabry, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporary+depletion+of+CD11c++dendritic+cells+delays+lymphoinvasion+after+intraperitonal+scrapie+infection.">Temporary depletion of CD11c+ dendritic cells delays lymphoinvasion after intraperitonal scrapie infection.</a> J Virol. 82, 8933-8933 (2008).</li><li>Dorban, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oral+scrapie+infection+modifies+the+homeostasis+of+Peyer's+patches'+dendritic+cells.">Oral scrapie infection modifies the homeostasis of Peyer's patches' dendritic cells.</a> Histochem Cell Biol. 128, 243-243 (2007).</li><li>Flores-Langarica, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scrapie+pathogenesis:+the+role+of+complement+C1q+in+scrapie+agent+uptake+by+conventional+dendritic+cells.">Scrapie pathogenesis: the role of complement C1q in scrapie agent uptake by conventional dendritic cells.</a> J Immunol. 182, 1305-1305 (2009).</li><li>Huang, F. P., MacPherson, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cells+and+oral+transmission+of+prion+diseases.">Dendritic cells and oral transmission of prion diseases.</a> Adv Drug Deliv Rev. 56, 901-901 (2004).</li><li>Ano, Y., Sakudo, A., Nakayama, H., Onodera, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uptake+and+dynamics+of+infectious+prion+protein+in+the+intestine.">Uptake and dynamics of infectious prion protein in the intestine.</a> Protein Pept Lett. 16, 247-247 (2009).</li><li>Aucouturier, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infected+splenic+dendritic+cells+are+sufficient+for+prion+transmission+to+the+CNS+in+mouse+scrapie.">Infected splenic dendritic cells are sufficient for prion transmission to the CNS in mouse scrapie.</a> J Clin Invest. 108, 703-703 (2001).</li><li>Huang, F. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Migrating+intestinal+dendritic+cells+transport+PrP(Sc)+from+the+gut.">Migrating intestinal dendritic cells transport PrP(Sc) from the gut.</a> J Gen Virol. 83, 267-267 (2002).</li><li>Jeffrey, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transportation+of+prion+protein+across+the+intestinal+mucosa+of+scrapiesusceptible+and+scrapie-resistant+sheep.">Transportation of prion protein across the intestinal mucosa of scrapiesusceptible and scrapie-resistant sheep.</a> J Pathol. 209, 4-4 (2006).</li><li>Raymond, C. R., Mabbott, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+the+involvement+of+migratory+dendritic+cells+in+the+transfer+of+the+scrapie+agent+from+the+immune+to+peripheral+nervous+systems.">Assessing the involvement of migratory dendritic cells in the transfer of the scrapie agent from the immune to peripheral nervous systems.</a> J Neuroimmunol. 187, 114-114 (2007).</li><li>Mohan, J., Hopkins, J., Mabbott, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin-derived+dendritic+cells+acquire+and+degrade+the+scrapie+agent+following+in+vitro+exposure.">Skin-derived dendritic cells acquire and degrade the scrapie agent following in vitro exposure.</a> Immunology. 116, 122-122 (2005).</li><li>Gilch, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CpG+and+LPS+can+interfere+negatively+with+prion+clearance+in+macrophage+and+microglial+cells.">CpG and LPS can interfere negatively with prion clearance in macrophage and microglial cells.</a> FEBS J. 274, 5834-5834 (2007).</li><li>Safar, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mass,+biochemical+composition,+and+physicochemical+behavior+of+the+infectious+form+of+the+scrapie+precursor+protein+monomer.">Molecular mass, biochemical composition, and physicochemical behavior of the infectious form of the scrapie precursor protein monomer.</a> Proc.Natl.Acad.Sci.U.S.A. 87, 6373-6373 (1990).</li><li>B&#252;eler, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mice+devoid+of+PrP+are+resistant+to+scrapie.">Mice devoid of PrP are resistant to scrapie.</a> Cell. 73, 1339-1339 (1993).</li></ol>

관심 없음 충돌 선언하지 않습니다.

여기 우리는 크게 말초 프리온 감염 초기에 이벤트를 모니터링 용이 생체내에 태그 및 추적 prions에 대한 프로토콜을 보여줍니다. 이 프로토콜은 크게 unambiguously 내생 PrP C에서 차별화에 사전 라벨 매우 풍부한 프리온 inoculum에 의해 체외 9 및 생체내 3,12의 모니터링 프리온 이해에서 과거 시도를 향상시킵니다. 전염성 prions와 PrP C가 동일한 기본...

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>CWD-infected elk brain</td>
<td>&nbsp;</td>
<td>Private elk farm in Colorado</td>
<td>&nbsp;</td>
<td>Use any non-human prion-infected brain</td>
</tr>
<tr>
<td>Blender</td>
<td>&nbsp;</td>
<td>Oster</td>
<td>6694-015</td>
<td>Use any commercial blender</td>
</tr>
<tr>
<td>Centrifuge</td>
<td>&nbsp;</td>
<td>Sorvall</td>
<td>SS34 rotor</td>
<td>Use any centrifuge /rotor that can reach 3000 x g and hold &ge; 500 ml volumes</td>
</tr>
<tr>
<td>Ultracentrifuge</td>
<td>&nbsp;</td>
<td>Beckman</td>
<td>50.2 Ti rotor</td>
<td>Use any ultracentrifuge /rotor that can reach 100,000 x g and hold &ge; 500 ml volumes</td>
</tr>
<tr>
<td>Bradford Reagent</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>B6916</td>
<td>&nbsp;</td>
<tr>
<td>Complete mini protease inhibitor cocktail</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>11 836 170 001</td>
<td>&nbsp;</td>
<tr>
<td>Sonicator</td>
<td>&nbsp;</td>
<td>Misonix</td>
<td>MP4000X</td>
<td>Use any horn or probe sonicator set to ~70% max power</td>
</tr>
<tr>
<td>DyLight antibody Labeling kit</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>53050</td>
<td>&nbsp;</td>
<tr>
<td>microcentrifuge</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>55430R</td>
<td>Use any refrigerated microcentrifuge that can achieve 13,000x g</td>
</tr>
<tr>
<td>centrifugal filter columns</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>Microcon YM-100</td>
<td>Use any filter or dialysis membrane with 100 Kd molecular weight cutoff</td>
</tr>
<tr>
<td>8-40 week-old FVB mice</td>
<td>&nbsp;</td>
<td>Charles River</td>
<td>207</td>
<td>Use any inbred mouse strain</td>
</tr>
<tr>
<td>1 &mu;m red fluorescent beads</td>
<td>&nbsp;</td>
<td>Phosphorex</td>
<td>2307</td>
<td>Use any fluorescent bead &le; 10 &mu;m</td>
</tr>
<tr>
<td>RPMI 1640 medium </td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11875-093</td>
<td>&nbsp;</td>
<tr>
<td>40 &mu;m cell strainer</td>
<td>&nbsp;</td>
<td>Falcon</td>
<td>352340</td>
<td>&nbsp;</td>
<tr>
<td>fluorescent antibodies</td>
<td>&nbsp;</td>
<td>BD pharmingen</td>
<td>Various</td>
<td>Use any fluorescent antibody appropriate for your application.</td>
</tr>
<tr>
<td>flow cytometer</td>
<td>&nbsp;</td>
<td>Dakocytomation</td>
<td>CyanADP</td>
<td>Use any flow cytometer capable of multicolor fluorescence detection</td>
</tr>		</tbody>
		</table>
		</div>

monitoring immune cells trafficking fluorescent prion rods hours after intraperitoneal infection

비정상적인 형태의 존재 프로 테아제, 모성 pathologic 및 전염성있는 호스트 인코딩된 프리온 단백질 (PrPC)는 같은 양의 cervids과 scrapie의 만성 낭비 질병 (CWD)와 같은 프리온 질병을 특징. 프리온 가설이 비정상 conformer가 대부분 또는 모든 감염성 프리온의를 종속 주장한다. 주변 프리온 pathogenesis 초기 행사에 면역 체계의 역할은 설득 CWD과 scrapie 1-3에 대한 증명되었습니다. 생쥐에 유전자 변형과 pharmacologic 연구는 감염 4-6 후 조기 prions을 유지하고 복제의 보완 시스템의 중요한 역할을 공개했다. 또한 70-10 돌기 세포 프리온 보존을 살펴 봤으니, 체외에서와 생체내 연구에서 매매에서 역할이 남아 있지만, 11-16 불분명. Macrophages는 마찬가지로 초기 프리온 pathogenesis에 연루되어 있지만,이 연구는 감염 후 3,11,17 주 발생하는 이벤트에 초점을 맞추고있다. 이러한 이전 연구는 또한 내생 PrP C와 전염성 prions 간의 차별의 문제에서 고통. 여기 우리가 모집 면역 세포에 의해 접종 사이트에서 프리온 이해와 인신 매매를 평가하기위한 semiquantitative, 편견 방식을 설명합니다. 집계 프리온 막대가 아닌 집계 단백질과 자당의 쿠션을 통해 ultracentrifugation의 세제 solubilization에 감염된 뇌 homogenate에서 정화했다. Polyacrylamide 젤 전기 영동, 쿠매시 푸른 얼룩과 pelleted 분수에 매우 풍부한 프리온 봉의 서쪽 모래 바닥 확인 회복. 프리온 막대는 마우스로 fluorochrome - 라벨이 다음 주입 intraperitoneally했다. 2 시간 후에 복막 세척 유체, 비장 및 mediastinal 및 mesenteric 림프절의 면역 세포는 프리온 막대기 유지 및 monocytes, neutrophils, 돌기 세포, macrophages 및 B 및 T 세포에 대한 마커를 사용하여 여러 가지 빛깔의의 유동세포계측법으로 식별 셀 집합에 대한 assayed했다. 이 분석은 감염 후 시간 이내에 면역 획득 세포와 생체내의 인신 매매 prions의 처음으로 직접 모니터링을 허용합니다. 이 분석은 또한 분명 어렵고 다른 분석 시스템의 데이터 해석에 문제가 발생할 수있는 일반적으로 호스트 세포에 표현 PrPC에서 전염성, 집계 prions를 차별화합니다. 이 프로토콜은 다른 접종 경로 (경구, 정맥, intranervous 및 피하 예)와뿐만 아니라 항원 (복합 비즈, 세균 바이러스 및 기생 병원균과 단백질, 계란)에 적용할 수 있습니다.

Watch this Scientific Journal Video about Monitoring Immune Cells Trafficking Fluorescent Prion Rods Hours after Intraperitoneal Infection at JoVE.com

Monitoring Immune Cells Trafficking Fluorescent Prion Rods Hours after Intraperitoneal Infection

여기 우리는 즉시 정화하여 intraperitoneal 접종 다음과 찬란 다음 주사 사이트에서 자신의 이해와 움직임을 모니터링하고 이러한 이벤트를 중재 전지 특성화 감염 두뇌 자료에서 집계 프리온 봉 라벨 면역 세포 프리온 이해와 인신 매매를 감시를위한 소설 분석을 설명합니다.

Intraperitoneal 감염 후 면역 세포의 인신 매매 형광등 프리온 로즈 시간을 모니터링

Presence of an abnormal form a host-encoded prion protein (PrPC) that is protease resistant, pathologic and infectious characterizes prion diseases such ...

monitoring-immune-cells-trafficking-fluorescent-prion-rods-hours

Research

JoVE Journal

Immunology and Infection

11.4K 조회수.  Colorado State University. 여기 우리는 즉시 정화하여 intraperitoneal 접종 다음과 찬란 다음 주사 사이트에서 자신의 이해와 움직임을 모니터링하고 이러한 이벤트를 중재 전지 특성화 감염 두뇌 자료에서 집계 프리온 봉 라벨 면역 세포 프리온 이해와 인신 매매를 감시를위한 소설 분석을 설명합니다.

비디오: Monitoring Immune Cells Trafficking Fluorescent Prion Rods Hours after Intraperitoneal Infection

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression 1. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis 1. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. 2 In this video, calpain activity in fixed and living murine 32Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32Dkit cells are shown to have elevated activity compared to normal splenocytes.

This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.

유동세포계측법로 고정하고 살아있는 세포에서 Calpain 활동을 측정

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

연구

면역학 및 감염병학

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

셀을 별 모양으로 일곱 단계

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam cells, immune cells, vascular endothelial and smooth muscle cells, platelets, extracellular matrix, and a lipid-rich core with extensive necrosis and fibrosis of surrounding tissues.1 The innate and adaptive arms of the immune response are involved in the initiation, development and persistence of atherosclerosis.2, 3 There is a significant body of evidence that different subsets of the immune cells, such as macrophages, dendritic cells, T and B lymphocytes, are present within the aortas of healthy and atherosclerosis-prone mice4. Additionally, immune cells are found in the surrounding aortic adventitia which suggests an important role of this tissue in atherogenesis.2
For some time, the quantitative detection of different types of immune cells, their activation status, and the cellular composition within the aortic wall was limited by RT-PCR and immunohistochemical methods for the study of atherosclerosis. Few attempts were made to perform flow cytometry using human aortas, and a number of problems, such as a high autofluorescence, have been reported5,6. Human atherosclerotic plaques were digested with collagenase 1, and free cells were collected and stained for CD14+/CD11c+ to highlight macrophage-derived foam cells. In this study, a "mock" channel was used to avoid false-positive staining.6 Necrotic materials accumulating during the digestion process give rise in a large amount of debris that generates a high autofluorescence in aortic samples. To resolve this problem, a panel of negative and positive controls has been proposed, but only double staining could be applied in these samples. We have developed a new flow cytometry-based method7 to analyze the immune cell composition and characterize the activation, proliferation, differentiation of immune cells in healthy and atherosclerosis-prone aorta. This method allows the investigation of the immune cell composition of the aortic wall and opens possibilities to use a broad spectrum of immunological methods for investigations of immune aspects of this disease.

This paper presents a flow cytometry-based method to investigate the immune composition of aortas. The paper also illustrates an additional technique that allows examining surrounding adventitia and vessel wall separately. This method opens possibilities to perform phenotypical analyses of aortic leukocytes and apply several immunological assays for atherosclerosis studies.

Murine Aortas 이내에 면역 세포의 흐름 Cytometry 분석

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

Isolation of Functional Cardiac Immune Cells

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5 These immune cells, which include mast cells, T-cells and macrophages; store and release a variety of biologically active mediators including cytokines and proteases such as tryptase.6-8 These mediators have been shown to be key players in extracellular matrix metabolism by activating matrix metalloproteinases or causing collagen accumulation by modulating the cardiac fibroblasts' function.9-11 However, available techniques for isolating cardiac immune cells have been problematic because they use bacterial collagenase to digest the myocardial tissue. This technique causes activation of the immune cells and thus a loss of function. For example, cardiac mast cells become significantly less responsive to compounds that cause degranulation.12 Therefore, we developed a technique that allows for the isolation of functional cardiac immune cells which would lead to a better understanding of the role of these cells in cardiac disease.13, 14
This method requires a familiarity with the anatomical location of the rat's xiphoid process, axilla and falciform ligament, and pericardium of the heart. These landmarks are important to increase success of the procedure and to ensure a higher yield of cardiac immune cells. These isolated cardiac immune cells can then be used for characterization of functionality, phenotype, maturity, and co-culture experiments with other cardiac cells to gain a better understanding of their interactions.

This method for isolating functional immune cells from the heart provides an alternative to the conventional methods of collagenase digestion, which causes unwanted immune cell activation, resulting in a decreased responsiveness of these cells. Our method of isolation yields functional cardiac immune cells by avoiding problems associated with enzymatic digestion.

심장 기능 면역 세포의 분리

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5  These immune cells, which ...

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

에 셀 운명을 따라<em&gt; E. 대장균</em&gt; 파지 람다에 의해 감염 후

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

기능 및 분자 연구 기본 손쥐 유형 II 폐포 상피 세포의 흐름 Cytometric 절연

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

Isolation of Adipose Tissue Immune Cells

The discovery of increased macrophage infiltration in the adipose tissue (AT) of obese rodents and humans has led to an intensification of interest in immune cell contribution to local and systemic insulin resistance. Isolation and quantification of different immune cell populations in lean and obese AT is now a commonly utilized technique in immunometabolism laboratories; yet extreme care must be taken both in stromal vascular cell isolation and in the flow cytometry analysis so that the data obtained is reliable and interpretable. In this video we demonstrate how to mince, digest, and isolate the immune cell-enriched stromal vascular fraction. Subsequently, we show how to antibody label macrophages and T lymphocytes and how to properly gate on them in flow cytometry experiments. Representative flow cytometry plots from low fat-fed lean and high fat-fed obese mice are provided. A critical element of this analysis is the use of antibodies that do not fluoresce in channels where AT macrophages are naturally autofluorescent, as well as the use of proper compensation controls.

Adipose tissue (AT) is a site of intense immune cell activation and interaction. Almost all cells of the immune system are present in AT and their ratios are altered by obesity. Proper isolation, quantification, and characterization of AT immune cell populations are critical for understanding their role in immunometabolic disease.

지방 조직 면역 세포의 분리

The discovery of increased macrophage infiltration in  the adipose tissue (AT) of obese rodents and humans has led to an  intensification of interest in ...

Generation of Lymph Node-fat Pad Chimeras for the Study of Lymph Node Stromal Cell Origin

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the mechanisms governing its formation. Lymph nodes are always encapsulated in adipose tissue and we recently demonstrated the importance of this relation for the formation of lymph node stroma. Adipocyte precursor cells migrate into the lymph node during its development and upon engagement of the Lymphotoxin-b receptor switch off adipogenesis and differentiate into lymphoid stromal cells (B&#233;n&#233;zech&#160;et al.14). Based on the lymphoid stroma potential of adipose tissue, we present a method using a lymph node/fat pad chimera that allows the lineage tracing of lymph node stromal cell precursors. We show how to isolate newborn lymph nodes and EYFP+ embryonic adipose tissue and make a LN/ EYFP+ fat pad chimera. After transfer under the kidney capsule of a host mouse, the lymph node incorporates local adipose tissue precursor cells and finishes its formation. Progeny analysis of EYFP+ fat pad cells in the resulting lymph nodes can be performed by flow-cytometric analysis of enzymatically digested lymph nodes or by immunofluorescence analysis of lymph nodes cryosections. By using fat pads from different knockout mouse models, this method will provide an efficient way of analyzing the origin of the different lymph node stromal cell populations.

Generation of lymph node/fat pad chimeras for the study of lymph node stromal cell origin is described. The method involves the isolation of lymph nodes from newborn mice and embryonic fat pads, the generation of chimeric lymph node-fat pads, and their transfer under the kidney capsule of a host mouse.

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the ...

Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable method of analysis by flow cytometry to study nanoparticle interaction with immune cells. Primary immune cells can be easily purified from human or mouse tissues by antibody-mediated magnetic isolation. In the first instance, the different cell populations running in a flow cytometer can be distinguished by the forward-scattered light (FSC), which is proportional to cell size, and the side-scattered light (SSC), related to cell internal complexity. Furthermore, fluorescently labeled antibodies against specific cell surface receptors permit the identification of several subpopulations within the same sample. Often, all these features vary when cells are boosted by external stimuli that change their physiological and morphological state. Here, 50 nm FITC-SiO2 nanoparticles are used as a model to identify the internalization of nanostructured materials in human blood immune cells. The cell fluorescence and side-scattered light increase after incubation with nanoparticles allowed us to define time and concentration dependence of nanoparticle-cell interaction. Moreover, such protocol can be extended to investigate Rhodamine-SiO2 nanoparticle interaction with primary microglia, the central nervous system resident immune cells, isolated from mutant mice that specifically express the Green Fluorescent Protein (GFP) in the monocyte/macrophage lineage. Finally, flow cytometry data related to nanoparticle internalization into the cells have been confirmed by confocal microscopy.

Analysis of nanoparticle interaction with defined subpopulations of immune cells by flow cytometry.

유동 세포 계측법에 의해 주요 면역 세포 모집단과 형광 나노 입자의 상호 작용의 검출

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable ...

A Semi-automated Approach to Preparing Antibody Cocktails for Immunophenotypic Analysis of Human Peripheral Blood

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease and treatment. It has the potential to predict therapeutic efficacy and identify novel therapeutic targets. Whole blood staining utilizes unmanipulated blood, which minimizes artifacts that can occur during sample preparation. However, whole blood staining must also be done on freshly collected blood to ensure the integrity of the sample. Additionally, it is best to prepare antibody cocktails on the same day to avoid potential instability of tandem-dyes and prevent reagent interaction between brilliant violet dyes. Therefore, whole blood staining requires careful standardization to control for intra and inter-experimental variability.
Here, we report deployment of an automated liquid handler equipped with a two-dimensional (2D) barcode reader into a standard process of making antibody cocktails for flow cytometry. Antibodies were transferred into 2D barcoded tubes arranged in a 96 well format and their contents compiled in a database. The liquid handler could then locate the source antibody vials by referencing antibody names within the database. Our method eliminated tedious coordination for positioning of source antibody tubes. It provided versatility allowing the user to easily change any number of details in the antibody dispensing process such as specific antibody to use, volume, and destination by modifying the database without rewriting the scripting in the software method for each assay.
A proof of concept experiment achieved outstanding inter and intra- assay precision, demonstrated by replicate preparation of an 11-color, 17-antibody flow cytometry assay. These methodologies increased overall throughput for flow cytometry assays and facilitated daily preparation of the complex antibody cocktails required for the detailed phenotypic characterization of freshly collected anticoagulated peripheral blood.

Whole blood Immunophenotyping is indispensable for monitoring the human immune system. However, the number of reagents required and the instability of specific fluorochromes in premixed cocktails necessitates daily reagent preparation. Here we show that semi-automated preparation of staining antibody cocktail helps establish reliable immunophenotyping by minimizing variability in reagent dispensing.

사람 말초 혈액의 Immunophenotypic 분석에 대한 항체 칵테일을 준비하는 반 자동화 된 접근

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease ...