The authors thank the Canadian Blood Services for a Graduate Fellowship Program Award to T.N.T. This research received financial support from the Canadian Blood Services' Centre for Innovation, funded by the federal government (Health Canada) and the provincial and territorial ministries of health. The views herein do not reflect the views of the federal, provincial, or territorial governments in Canada.

연구 윤리 보드 / 기관 검토위원회에 의해 인간의 샘플의 사용에 대한 승인을 받아야하고, 모든 사람의 기증자로부터 서명 동의를 구하십시오. MMA를 또한 사용 동물 윤리 승인에 따라 인간의 분석과 동일한 방법으로 마우스 PBMC를 사용하여 수행 될 수있다. MMA의 무균 조직 배양 기술을 활용한다. 주 : 그림 1을 참조하십시오. 참고 :이 방법은 단지 &quot;단기&quot;배양 방법이면, 무균 기술을 유지하기 위해 분석하는 동안 레벨이 biocontainment 캐비닛의 사용을 권장하지만, 세균 분석을 오염 정말로 시간이 충분하지 않다. 레벨 2 biocontainment 캐비닛 존재보다는이 분석을 위해 하나를 구입하지 않는 경우 따라서 분석은 오픈 벤치에하는 biocontainment 캐비닛의 외부에서 수행 될 수있다. 5 % CO 2, 37 ° C의 배양기를 사용하지만 옵션 아니며 사용되어야최적의 결과. 1. 말초 혈액 단핵 세포 분리 <ol><li> 건강한 기증자 또는 산 시트 레이트 - 덱스 트로 오스 (ACD) 항응고제 (노란색 탑 튜브)를 포함 vacutainers를 사용하여 정맥 천자를 통해 환자로부터 인간의 전체 혈액을 얻습니다. 주 : 전혈 다음 단계 (11)로 진행하기 전에 36 시간 동안 실온 (18 ~ 22 °의 C)에서 ACD에 저장 될 수있다. 일반적 전혈 1 ~ 10 ML의 vacutainer 튜브 분석에 충분하다. </li><li> 전혈 희석 1 : 1을 V / (10 % 소 태아 혈청, 20 mM의 HEPES, 0.01 ㎎ / ㎖ 젠타 마이신으로 보충 된 RPMI-1640) 따뜻한 완전 RPMI 배지에서 V. </li><li> 제조자가 권장하는 밀도 구배 원심 분리를 사용하여 희석 된 전혈로부터 말초 혈액 단핵 세포 (PBMC)를 분리 (재료의 목록 참조). 밀도 구배에 걸쳐 매우 천천히 희석 혈액 레이어 (실온, 18 ~ 22 ℃로 가온). 참고 믹시의 양을 최소화조심스럽게 적하 방식으로 혈액 혼합물을 적층하여 또는 피펫을 사용하여 혈액의 최적 분리를위한 인터페이스에 ng를. <ol><li> 혈액 혼합물이 천천히 밀도 구배에 가까운 피펫 팁을 배치함으로써, 매우 천천히 관의 측면을 실행하는 피 혼합물을 활성화하여 농도 구배의 맨 위에 레이어 할 수있다. </li><li> 50 mL의 실험 규모 층 혈액 (15 ㎖의 튜브에서) 밀도 구배의 3 ㎖의 위에 혼합물 또는 층 밀도 구배 (15 ㎖의 위에 피 액 35 mL를 10 ㎖에 따라 튜브). 통상적으로, 전체 혈액 10 ㎖의 일부 도너 투 도너 변화량 천만 한 PBMC를 산출한다. 주 : 밀도 구배와 혈액 혼합물의 믹싱이없는 것이 매우 중요합니다. 혈액 혼합물을 밀도 구배 위에 층 전체 혈액 농도 구배 위에있을 때까지 천천히 상승한다. </li><li> 브레이크없이 30 분 동안 700 XG에 적층 된 혼합물을 원심 분리기. CENTR혈장, 버피 코트 (PBMC를 함유), 밀도 구배 물질, 과립구, 및 적혈구 : ifuged 혼합물 (위에서 아래로) 5 층으로 분리한다. </li><li> 삭제 플라즈마의 대부분을 폐기하고 신중 파스퇴르 피펫 및 흡입 벌브를 사용하여 새 15-㎖의 튜브에 버피 코트 (PBMC)의 컨텐츠를 검색한다. 주 : 버피 코트 층의 제거를 효과적으로 층의 외측 주위에 원 운동을 수행하는 동안 상기 튜브에 대한 피펫의 팁의 파스퇴르 피펫에 흡입을 적용함으로써 수행된다. </li></ol></li><li> 세척 사이에서 (풀 브레이크) 10 분 동안 350 × g으로 원심 분리에 의해 단리 한 PBMC를 7.3 pH를 인산 완충 용액 (PBS)에 세 번 세척 하였다. 완전 RPMI 배지에서 PBMC 펠릿을 재구성한다. 펠렛의 크기에 따라, 매체의 3-7 mL로 충분하다. </li><li> 트리 판 블루와 혈구를 사용하여 PBMC를 계산합니다. 만 트리 판 블루로 염색되지 않은 그 세포를 계산합니다. Reconstitut전체 RPMI 배지에서 1,750,000 세포 / ㎖로 전자 PBMC를. </li><li> 8 챔버 슬라이드의 각 웰에 400 μL 시드 (700,000 세포). 37 ° C, 완전히 습윤 조직 배양 인큐베이터에서 인큐베이션 슬라이드 단핵구 / 대 식세포를 부착 할 수 있도록 한 시간 동안 (5 % CO 2로 보충). </li></ol> 첨착 단핵구 2. 전처리 참고 : 식균 작용을 억제하거나 강화하는 방법을 찾고 있다면이 단계는 필요하다. <ol><li> 사전 치료는 관심의 약물 (들) 또는 화합물 (들)와 단핵구를 부착. 완전한 RPMI 배지를 사용하여 원하는 농도로 약물 (들) 또는 다른 시험 물질을 재구성한다. 참고 : 예를 들어, 200 μg의 IVIG / ㎖ 전형적 인간 단핵 세포를 사용하는 경우의 탐식 95~100% 억제를 수득하기 위해 사용된다. </li><li> 대기음 (단계 160 후) 1 시간 배양 한 후 8 챔버 슬라이드에서 비 부착 세포를 함유하는 상등액을 버리고. (400)로 교체약물이나 다른 치료 μL, 37 ° C에서 1 시간 동안 품어. <ol><li> 흡입 및 8 챔버 슬라이드에 솔루션을 교체 할 때, 약하게 부착 된 세포가 이륙하지 않도록 유체의 흐름을 제어해야합니다. 또한, 한 번에 2 ~ 3 빈 우물 작업과 우물을 건조하지 마십시오. 참고 : 일반적으로, 각 치료 기술 삼중으로 수행된다. </li></ol></li></ol> R 3. 옵 소닌 2 R 2 붉은 혈액 세포 참고 : 여기에 사용 된 R 2 R 2는 혈액 수집 센터 (캐나다 혈액 서비스)에서 얻을 수 있습니다,하지만 그들은 또한 시판되고있다. 옵 소닌 R 2 R 2 적혈구는 FcγR 매개 식균 작용을위한 양성 대조군으로 사용된다. 순진 R 2 R이 1 달까지 4 ° C에서 Alsever의 솔루션 (연장하기 위해 수명)에 저장해야합니다. Alsever의 솔루션은 사내에서 만든 0.8로 구성되어있다% / V 트리 나트륨 시트 레이트 (수화물) 1.9 % w / v를 덱 스트로스, 0.42 % w / v를 염화나트륨 및 0.05 % w / v를 시트르산 (수화물) w R 2 R 경우이 세포는 R 1 R 2, R 1 R 1, R 1, R 또는 R 2 R, 사용할 수있는 다른 RH-phenotyped 세포는 사용할 수 없습니다. <ol><li> 워시 R 2 R 2 (CDE / CDE) PBS 적혈구 5 분마다 350 XG 원심 분리를 3 회 총. 주 : 필요한 적혈구의 양이 실험 규모에 따라 다시 계산 될 수있다. 적혈구 인해 용해 또는 상층 액을 제거하는 동안 각각의 세척 중에 손실되기 때문에 항상, 적혈구의 과량을 씻는다. 예를 들어, 기술 3 회 반복 실시 (5) 치료와 두 컨트롤 모두와 함께 실험에서, 21 우물의 총이있다. 400 μL / 웰의 1.25 % RBC 혼합물 21 우물은 포장 된 105 μL가, RBC이 필요 옵 소닌을 나타냅니다. (200) RBC의 μL 처음에 세척해야하며, 150 &amp; #181; L은 식균 작용 단계에 대한 적혈구 적당량가되도록하여 옵 소닌 화되어야한다. </li><li> 1 세척 R 2 R 2 펠렛 Opsonize : 사람 혈청에서 1 v / V를 클론 항 D 항체를 간헐적 혼합하여, 37 ℃에서 1 시간 동안 배양. 주 : 다 클론 항 D 항체를 사용할 수없는 경우는 시판되고있는 단일 클론 항 D 항체 또는 혈액 은행 또는 수혈에서 얻을 수 통상의 Rh 면역 예방에 사용되는 항 - D를 사용하는 것이 가능하다 서비스. 분석을 설정할 때, 그리고 비 - 역가 클론 항체의 많은 스위칭마다 또는 모노클로 날 항체로 전환 최적화 실험은 초기에 수행되어야한다. 이것은 글로불린 시험 3+ 사이 4+ 및 계산 (100) 단핵 세포 당 70 ~ 90 사이의 포식 적혈구의 식균 작용의 결과 (IAT) 결과에 필요한 최적의 농도 또는 항-D의 볼륨을 확인하는 것입니다. </li><li> opsonize을 씻으십시오D의 R이 R이 각각 5 분, 350 × g으로 원심 분리를 이용하여 PBS에서 3 회 전체. 주 : R이 R이 성공적으로 옵 소닌 간접 IAT를 수행하여 확인할 수있다. 간략하게, 이차 클론 항 - 인간 항체 RBC 표면 상 opsonizing 일차 항체에 결합하기 위해 추가되고, 증폭 된 신호는 혈구 응집의 형태로 관찰 할 수있다. 상세한 제조 업체 프로토콜은 보충 자료 파일에서 찾을 수 있습니다. </li><li> 1.25 % v / V를 완료 RPMI 매체를 사용하여 세척 R 2 R 2 펠렛을 복원합니다. 주 : 옵 소닌 잉여 R이 R 2는 주까지 4 ℃에서 Alsever의 용액에 저장 될 수있다. </li></ol> 4. Fc 수용체 - 매개 탐식 <ol><li> 8 챔버 슬라이드에서 약물 또는 중간 뜨는을 기음과 1.25 %의 V / V의 R 2 R 2 혼합물의 400 μL를 추가합니다. 2 시간 동안 37 ℃에서 인큐베이션. </li><li> 선미어 배양 2 시간은 제조업체의 어댑터를 사용하여 챔버를 제거합니다. 종이 타월에 과잉 R 2 R 2를 DAB. 슬라이드가 건조하지 않도록해야합니다. </li><li> PBS와 100 mL의 비커를 입력합니다. 잠수함 천천히 해제 포식 R 2 R (2)의 대부분을 제거하기 전후 (30-40 선 주위)를 슬라이드 이동시킴으로써 슬라이드를 세척한다. </li><li> PBS를에서 슬라이드를 제거합니다. 종이 타월 또는 조직 및 사용 초과 PBS 오프 DAB 슬라이드를 공기 - 건조. </li><li> 45 초 동안 100 % 메탄올의 공기 건조 슬라이드를 수정합니다. 그런 다음, 고정 된 슬라이드를 공기 - 건조. 참고 : 슬라이드 같은 그룬 왈드 - 김사 염색 (21) 또는 라이트 - 김사 염색 (22)로, 하류 염색에 더 호환되는 다른 방법을 사용하여 고정 할 수 있습니다. </li><li> elvanol 설치 매체 (또는 다른 시판 설치 중) 집에서 만든-및 추가 된 커버를 사용하여 슬라이드를 탑재합니다. 참고 : Elvanol 설치 매체 / V 포 w는 15 %로 구성되어있다PBS에서 lyvinyl 알콜 수지 30 % v / V를 글리세린. 모든 수지가 용해 될 때까지 혼합물을 가열하고, 글리세린이 균일하게 혼합한다. 이것은 다른 시중에서 판매하는 설치 매체로 대체 될 수있다. </li><li> 마운트가 정량화하기 전에 밤새 건조하도록 허용합니다. </li></ol> 탐식의 5 정량화 <ol><li> 위상 현미경 및 40X 대물 렌즈를 이용하여 수동으로 적어도 200 단핵구 이들 단핵 포식 내의 R 2 R (2)의 수를 카운트하여 탐식 이벤트의 양을 정량화. 동시에 단핵 세포의 수와 포식 R 2 R 2의 수를 정량화하기 위해 각 손에 하나의 카운터가 있습니다. </li><li> 단핵 세포의 수에 의해 포식 R 2 R (2)의 분할 수 (100) 및 고속 평균 (SEM)의 ± 표준 오차의 평균으로서 데이터 (평균 식세포 인덱스)을 곱하여 평균 식세포 인덱스를 얻었다. </li&gt; </ol>

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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+papain+on+the+interaction+between+human+monocytes,+erythrocytes+and+IgG.">Effect of papain on the interaction between human monocytes, erythrocytes and IgG.</a> Blood. 46 (2), 245-252 (1975).</li><li>Tong, T. N., 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=Optimal+conditions+for+the+performance+of+a+monocyte+monolayer+assay.">Optimal conditions for the performance of a monocyte monolayer assay.</a> Transfusion. , (2016).</li><li>Gray, S. J., Sterling, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tagging+of+red+cells+and+plasma+proteins+with+radioactive+chromium.">The tagging of red cells and plasma proteins with radioactive chromium.</a> J Clin Invest. 29 (12), 1604-1613 (1950).</li><li>Mollison, P. L., Veall, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+the+isotope+51Cr+as+a+label+for+red+cells.">The use of the isotope 51Cr as a label for red cells.</a> Br J Haematol. 1 (1), 62-74 (1955).</li><li>Sebring, E. S., Polesky, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+fetal+hemorrhage+in+Rh+immune+globulin+candidates.+A+rosetting+technique+using+enzyme-treated+Rh2Rh2+indicator+erythrocytes.">Detection of fetal hemorrhage in Rh immune globulin candidates. A rosetting technique using enzyme-treated Rh2Rh2 indicator erythrocytes.</a> Transfusion. 22 (6), 468-471 (1982).</li><li>Downing, I., Templeton, J. G., Mitchell, R., Fraser, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chemiluminescence+assay+for+erythrophagocytosis.">A chemiluminescence assay for erythrophagocytosis.</a> J Biolumin Chemilumin. 5 (4), 243-250 (1990).</li><li>Fabron, 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=Application+of+noninvasive+phagocytic+cellular+assays+using+autologous+monocytes+to+assess+red+cell+alloantibodies+in+sickle+cell+patients.">Application of noninvasive phagocytic cellular assays using autologous monocytes to assess red cell alloantibodies in sickle cell patients.</a> Transfus Apher Sci. 31 (1), 29-35 (2004).</li><li>Michelis, F. V., 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=Acute+hemolysis+after+intravenous+immunoglobulin+amid+host+factors+of+ABO-mismatched+bone+marrow+transplantation,+inflammation,+and+activated+mononuclear+phagocytes.">Acute hemolysis after intravenous immunoglobulin amid host factors of ABO-mismatched bone marrow transplantation, inflammation, and activated mononuclear phagocytes.</a> Transfusion. 54 (3), 681-690 (2014).</li><li>Rampersad, G. 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=Chemical+compounds+that+target+thiol-disulfide+groups+on+mononuclear+phagocytes+inhibit+immune+mediated+phagocytosis+of+red+blood+cells.">Chemical compounds that target thiol-disulfide groups on mononuclear phagocytes inhibit immune mediated phagocytosis of red blood cells.</a> Transfusion. 45 (3), 384-393 (2005).</li><li>Purohit, M. K., 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=Structure-activity+relationships+of+pyrazole+derivatives+as+potential+therapeutics+for+immune+thrombocytopenias.">Structure-activity relationships of pyrazole derivatives as potential therapeutics for immune thrombocytopenias.</a> Bioorg Med Chem. 22 (9), 2739-2752 (2014).</li><li>Neschadim, A., Kotra, L. P., Branch, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+molecule+phagocytosis+inhibitors+for+immune+cytopenias.">Small molecule phagocytosis inhibitors for immune cytopenias.</a> Autoimmun Rev. 15 (8), 843-847 (2016).</li><li>Fitzer-Attas, C. J., 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=Fcgamma+receptor-mediated+phagocytosis+in+macrophages+lacking+the+Src+family+tyrosine+kinases+Hck,+Fgr,+and+Lyn.">Fcgamma receptor-mediated phagocytosis in macrophages lacking the Src family tyrosine kinases Hck, Fgr, and Lyn.</a> J. Exp Med. 191 (4), 669-682 (2000).</li><li>Allhorn, 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=The+IgG-specific+endoglycosidase+EndoS+inhibits+both+cellular+and+complement-mediated+autoimmune+hemolysis.">The IgG-specific endoglycosidase EndoS inhibits both cellular and complement-mediated autoimmune hemolysis.</a> Blood. 115 (24), 5080-5088 (2010).</li><li>Li, L., 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=Inhibition+of+phagocytic+recognition+of+anti-D+opsonized+Rh+D++RBC+by+polymer-mediate+immunocamouflage.">Inhibition of phagocytic recognition of anti-D opsonized Rh D+ RBC by polymer-mediate immunocamouflage.</a> Am J Hematol. 90 (12), 1165-1170 (2015).</li><li>Kwan, D. 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=Toward+efficient+enzymes+for+the+generation+of+universal+blood+through+structure-guided+directed+evolution.">Toward efficient enzymes for the generation of universal blood through structure-guided directed evolution.</a> J Am Chem Soc. 137 (17), 5695-5705 (2015).</li></ol>

The authors have nothing to disclose.

MMA의 조직 문화와 현미경 모두에 대한 전문 지식을 필요로하는 힘든 기술이다. 성공을 보장하기 위해 몇 가지 중요한 단계가 다음과 같습니다 단핵 세포 단층의 1) 생성; 적혈구의 2) 옵 소닌, 3) 매뉴얼 정량화. 단핵구 단층은 상기 챔버 슬라이드에 강력하게 부착되지 않기 때문에, 생리 학적 pH를 11 시험 법에 걸쳐 유지되어야하며, PBMC를 적절한 수는 배정되어야한다. 부착 된 세포를 방해 할 수 활발한 피펫은 피해야한다. 하나의 방법은 항상 제거 챔버의 동일한 코너의 솔루션을 추가하고, 움직임이 느리고 정상인지 확인한다. 마찬가지로, 여분의 적혈구를 제거하는 마지막 세척 단계 동안, 운동은 천천히 그리고 꾸준히해야한다. 아직도 않은 포식 적혈구의 대부분을 제거하는 동안이 단층에 최소한의 교란을 보장합니다. 불충분 한 세척 수동 ...로서하게 오염 적혈구의 높은 배경으로 이어질 것입니다ntification 어렵다. 둘째, R 2 R이 적혈구 충분히 식세포 제어 80-120 평균 식세포 인덱스를 얻기 위해 옵 소닌 화되어야한다. 이것은 원하는 식세포 범위 계산의 용이성 간의 균형을 통계 분석 식세포 적당량 유지 (예를 들어, 5 개 이상으로 단핵구는 적혈구 정확하게 정량화하기 어렵다 포식). 옵 소닌 화의 정도는 IAT에 의해 확인 될 수 있고, 4 이상 3+ 사이의 판독이 필요하다. 이 적혈구 거기 상등액 암적색 바뀌면 세정 동안 과도한 용해 때 또는 폐기되어야하는 R이 R 식세포의 상당한 감소로 인해 스토리지 세포의 노화 실험에서 관찰되는 경우. 실험실 직원 사이의 실험 사이의 수를 비교 특히 마지막으로, 현미경을 사용하여 수동 정량화는 까다로울 수있다. 각 웰에 동일한 필드를 검사함으로써, 또는 단순히 이상의 셀을 계산하여좀더 일관 카운트를 얻을 수있다. 숙련 된 기술자 및 교육 슬라이드 지정된 세트의 사용과 나란히 훈련하는 것이 좋습니다. MMA의 주요 비판 수동 정량화 단계의 주관적이다. 그러나, 적절한 교육과, 일관성은 서로 다른 카운터를 통해 얻을 수있다. 또 다른 한계는 인간 시료를 처리 데이터의 변화의 원인이고 R이 2 표면 항원을 발현 수준, 단핵 식세포 능력 및 R의 고유 도너 투 도너 차이이다. 다른 대안 기술은 FcγR 매개 식균 작용을 검사 할 수 있습니다. 상업용 키트 대부분은 식균 작용을 모니터링하는 형광 출력을 이용 (예 bioparticles, pH 민감성 형광 단백질, 또는 IgG에 표지 된 형광 라텍스 비즈). 형광 출력의 사용은보다 객관적인 정량화를 제공 않지만, 하나는 사기 필요형광 현미경 유세포뿐만 아니라, 시판 키트에 따라 계속되는 의존성의 사용과 관련된 사용 가능성, 비용 및 교육을 생각 해보자. 마지막으로, 상기 분석은 연구 문제에 따라 변경 될 수있다. 식세포 작용의 억제 약물을 시험 할 때 예를 들어, 단핵구는 사전 처리 될 수 있거나, 약물과 적혈구 옵 소닌 화 (즉, 경쟁 분석) 모두는 공동 - 배양 하였다. 다른 서브 타입, 키메라 항체 또는 재조합 항체의 구조의 다운 스트림 신호는 테스트 될 수있다. 보편적 인 항원 - 널 피 (24)의 개발에 최근 돌파구로, MMA는 식균 작용을 유발의 감소 효과가 실제로 존재 여부를 평가하기 위해 다양한 항체 이러한 항원 널 적혈구의 초기 화면에 이용 될 수있다.

The monocyte monolayer assay (MMA) is an in vitro assay originally developed to better predict blood transfusion outcomes in patients with auto- or alloantibodies to red blood cells (RBCs)1-5. By assessing the effect of anti-RBC antibodies in mediating Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis using this in vitro assay, it is possible to predict the clinical outcome in vivo. Indeed, the MMA has been used successfully to avoid immune destruction of antibody-bound RBCs, despite the transfusion of serologically incompatible blood5. The typical pre-transfusion procedure for compatibility testing, also termed crossmatching, involves serological methods that include typing the patient's blood for ABO and Rh antigens and screening for the presence of anti-RBC antibodies in the patient6. Blood matched for ABO/Rh is selected, and if antibodies are present, an attempt to identify them is made so that blood for transfusion can be further selected to avoid these antigens. An ideal crossmatch result occurs when all donor blood is serologically compatible with the patient's blood, which reduces the risk of post-transfusion hemolysis7. However, this system falls short for the small group of patients who have become alloimmunized upon repeated transfusion or pregnancy. These patients produce alloantibodies against specific RBC antigens. Some produce antibodies to antigens of very high frequency in the general population, and thus become progressively more difficult to crossmatch8,9. Adding to the complexity, not all alloantibodies are clinically significant; in other words, the binding of an alloantibody to RBCs detected by a serology test does not necessarily result in hemolysis when antigen-positive, incompatible blood is transfused. The MMA was originally developed to assess the potential clinical significance of serologically incompatible blood in a transfusion setting1-5.
Since extravascular hemolysis of antibody-bound RBCs is known to be mediated by the mononuclear phagocyte system, primary monocytes/macrophages are utilized in the development of diagnostic assays. The first assay to study the interaction of monocytes, RBCs, and antibodies was published in 1975, but the sub-optimal conditions used led only to rosette formation (the binding of RBCs to the periphery of the monocyte), and no phagocytosis was observed10. Significant modifications to the assay were made by several groups, leading to an assay for which the level of phagocytosis of alloantibody-bound RBCs could be correlated to the clinical outcome of hemolysis1-5. Recently, the optimal storage conditions of clinical samples and further optimization of assay conditions were examined to enhance the utility of a clinical MMA crossmatch using autologous patient samples11.
Three other diagnostic techniques have been employed in addition to the MMA in predicting transfusion outcomes: the 51Cr release test, the rosette test, and the chemiluminescence test (CLT). In the 51Cr release test, the patient is injected with 51Cr-labeled donor RBCs, and the half-life of the labeled RBCs is monitored and is predictive of post-transfusion survival or clearance12,13. As this method uses radioactive materials, it is rarely performed anymore. The rosette test involves mixing and incubating monocytes with RBCs and quantifying the level of rosette formation (with no phagocytosis)14. The clinical significance of antibodies in vivo involves active phagocytosis by macrophages found in the spleen and/or the liver; thus, this method does not provide a relevant readout of phagocytosis. The CLT uses luminol to monitor the oxidative burst during monocyte phagocytosis of RBC, since luminol fluoresces blue when oxidized in the phagosome15. This method is good, but contamination by neutrophils can confound the readout. Parallel comparisons have been made to evaluate the sensitivity, practicality, and reproducibility of the four available methods, and both the CLT and MMA were ranked superior16. However, the CLT has been mainly utilized in assessing hemolytic disease of the fetus and newborn (HDFN), and the assay's optimal pH of 8.0 might compromise the level of phagocytosis11.
In addition to its diagnostic and clinical utility, the MMA has been modified for other research purposes. Indeed, the MMA can not only serve as a functional assay to address discrepancies between serology and biology, it has also been used to retrospectively investigate the cause of hemolysis after intravenous immunoglobulin (IVIG) therapy17. It has also been used to examine the structure-function of chemical inhibitors of Fc&#947;R-mediated phagocytosis18-20 and to study the downstream signaling of Fc&#947;R-mediated phagocytosis21. In our laboratory, in addition to using a human MMA, we are developing a murine MMA using primary mouse peripheral blood mononuclear cells (PBMCs) and autologous RBCs. The rationale is to screen antibodies that can induce Fc&#947;R-mediated phagocytosis as an intermediate to developing an in vivo autoimmune hemolytic anemia (AIHA) mouse model (unpublished data). The various modifications focus on different aspects of the IgG antibody and Fc&#947;R interaction that induce phagocytosis.

<table border="0" cellpadding="0" cellspacing="0" width="1148">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Acid citrate dextrose (ACD) vacutainers</td>
			<td style="width:213px;">BD</td>
			<td style="width:136px;">REF364606</td>
			<td style="width:405px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">RPMI 1640</td>
			<td style="width:213px;">Sigma</td>
			<td style="width:136px;">R8758</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">HEPES</td>
			<td>Bioshop</td>
			<td>HEP003.100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Fetal bovine serum</td>
			<td style="width:213px;">Multicell&#160;</td>
			<td style="width:136px;">080150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Gentamicin</td>
			<td>Gibco</td>
			<td>15710-64</td>
			<td></td>
		</tr>
		<tr height="100">
			<td height="100" style="height:100px;width:395px;">Ficoll-Paque PLUS</td>
			<td style="width:213px;">GE</td>
			<td style="width:136px;">17-1440-03</td>
			<td><a href="https://www.gelifesciences.com/">https://www.gelifesciences.com/</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Phosphate buffered saline</td>
			<td style="width:213px;">Sigma</td>
			<td style="width:136px;">D8537</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">8-chamber slides</td>
			<td style="width:213px;">Lab-Tek-ll</td>
			<td style="width:136px;">154534</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:395px;">R2R2 (cDE/cDE) red blood cells&#160;</td>
			<td style="width:213px;">Canadian Blood Services</td>
			<td style="width:136px;"></td>
			<td>Commercially available (e.g. http://www.bio-rad.com/en-ca/product/reagent-red-blood-cells)&#160;</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:395px;">Polyclonal anti-D from human serum</td>
			<td>Gamma Biologics</td>
			<td>DIN 02247724</td>
			<td>Can be substituted with commercially available monoclonal anti-D or with Rh immune globulin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">100% methanol</td>
			<td style="width:213px;">Caledon</td>
			<td style="width:136px;">6700-1-42</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Polyvinyl alcohol resin</td>
			<td style="width:213px;">Sigma</td>
			<td style="width:136px;">P8136</td>
			<td>Can be substituted with commercially available mount</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">UltraPure glycerine</td>
			<td style="width:213px;">Invitrogen</td>
			<td style="width:136px;">15514-011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Cover slips</td>
			<td style="width:213px;">VWR</td>
			<td style="width:136px;">48366 067</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Novaclone anti-IgG</td>
			<td style="width:213px;">Immucorgamma</td>
			<td style="width:136px;">5461023</td>
			<td>Optional for IAT (http://www.fda.gov/downloads/biologicsbloodvaccines/.../ucm081743.pdf)</td>
		</tr>
	</tbody>
</table>

use of a monocyte monolayer assay to evaluate fc&#947; receptor-mediated phagocytosis

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly versatile in vitro assay that can be modified to examine different aspects of antibody and Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis in both research and clinical settings. The assay utilizes adherent monocytes from peripheral blood mononuclear cells isolated from mammalian whole blood. MMA has been described for use in both human and murine investigations. These monocytes express Fc&#947;Rs (e.g., Fc&#947;RI, Fc&#947;RIIA, Fc&#947;RIIB, and Fc&#947;RIIIA) that are involved in immune responses. The MMA exploits the mechanism of Fc&#947;R-mediated interactions, phagocytosis in particular, where antibody-sensitized red blood cells (RBCs) adhere to and/or activate Fc&#947;Rs and are subsequently phagocytosed by the monocytes. In vivo, primarily tissue macrophages found in the spleen and liver carry out Fc&#947;R-mediated phagocytosis of antibody-opsonized RBCs, causing extravascular hemolysis. By evaluating the level of phagocytosis using the MMA, different aspects of the in vivo Fc&#947;R-mediated process can be investigated. Some applications of the MMA include predicting the clinical relevance of allo- or autoantibodies in a transfusion setting, assessing candidate drugs that promote or inhibit phagocytosis, and combining the assay with fluorescent microscopy or traditional Western immunoblotting to investigate the downstream signaling effects of Fc&#947;R-engaging drugs or antibodies. Some limitations include the laboriousness of this technique, which takes a full day from start to finish, and the requirement of research ethics approval in order to work with mammalian blood. However, with diligence and adequate training, the MMA results can be obtained within a 24-h turnover time.

그림 1 위에 설명 된 절차의 중요한 단계를 수행함으로써, MMA는 재현성을 수행 할 수 있습니다. IVIG는도 2의 식균 작용을 억제하기위한 예로서 사용 하였다. IVIG 따라서 식세포의 하류 결과를 억제 결합 및 Fc 수용체를 차단하는 것으로 알려져있다. 사용 IVIG의 양을 적정에 의해 용량 의존적 억제하는 / ml의 전체 (도 2A)에서 억제하지 않았다 / ㎖ 0.5 μg의 아래에 100 % 억제 가까이 농도 결과 200 μg의 이상의 농도가 관찰된다. 탐식 지수 변환 및 0 % 억제 / ㎖가 결정될 수 3 μg의의 IC (50) (도 2b)를 억제 곡선으로서 R이 R이 양성 대조군으로 정규화하는 경우. 지속적으로 분석을 수행하는 것 외에도 quantifica분석의 능은 때때로 어려울 수있다. 도 3은 여러 MMA 슬라이드 위상차 현미경 이미지의 집합이다. 현미경의 경험을 바탕으로, 하나는 오염 RBC (그림 3D), 또는 포식 RBC (그림 3B)에서 공포에서 단핵 세포를 구분할 수 있습니다. 세포 및 / 또는 파편의 고밀도 클러스터는 정량 (도 3E 및 F) 동안 피해야한다. 또한, 하나는 피해야 오버 opsonizing 단핵구 인테리어 overcrowds하고 정확한 정량 (그림 3C)을 방해 높게 식균 작용으로 이어질 것 R 2 R 2 RBC를. <img alt="그림 1" src="/files/ftp_upload/55039/55039fig1.jpg" /> 병무청의 도식도를 그림. 병무청에서 중요한 단계의 단계별 묘사 : 전체 혈액에서 PBMC를 분리, 부착 monocyt 공급옵 소닌 R 2 R 2 챔버 슬라이드를 세정하고 에스. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55039/55039fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 2" src="/files/ftp_upload/55039/55039fig2.jpg" /> 도 2 정맥의 IgG (IVIG)는 MMA를 사용하여 시험 관내 식세포 작용을 억제한다. IVIG는 식균 작용을 억제하는 것으로 알려져 있으며, 인간의 MMA 억제 제어부로서 사용된다. 미처리 R 2 R이 제어에 비해 IVIG의 (A)의 적정 식세포 용량 의존적 억제 결과. 결과의 평균, N = 3의 실험 (SEM)의 ± 표준 오차의 평균을 나타낸다. 통계 분석은 학생 t의 -test를 사용 하였다 : ** P≤0.01 및 *** P≤0.001. (B)는 IC (50) 오와 IVIG의 억제 곡선F 3 μg의 / ㎖ (점선). 결과는 치료 R 2 R 2 (0 % 억제)에 정규화 데이터를 표시; N = 3 실험 ± SEM을 의미한다. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55039/55039fig2large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 3" src="/files/ftp_upload/55039/55039fig3.jpg" /> 40X 배율에서 샘플 슬라이드 그림 3. 위상차 현미경 이미지. (A)을 단핵구가 배경 (흰색 화살표)에 거의 오염, 취소 포식 R 2 R 2, 평균 (특유의 후광 빛이 함께 검은 색 화살표)에 1-2 R 2 R 2를 포식하는 완벽한 슬라이드. 포식 R 2 R이 오인 될 수있는이 슬라이드 (b)에 나타낸 바와 같이, 단핵구는 때때로 확대 한 액포 (흰색 화살표). (C &lt;/ strong&gt;을) 이상 4-5 R 2 단핵구 당 R 2 (검은 색 화살표를 포식, 옵 소닌을 통해-R 2 R 2가되었을 때)가 R 2 R 2는 단핵구와 별개의 셀 내에서 혼잡하기 때문에 정확하고 어려운 계산 렌더링 경계는 구별 할 수 없습니다. (D) 슬라이드의 불충분 한 세척이 부착 된 적혈구 오인 될 수있는 풍부한 R 2 R 2 오염 (흰색 화살표)로 연결됩니다. (E) 때때로, 단핵구 및 오염 적혈구가 클러스터를 형성 할 수있다. 클러스터는 피해야한다 세균 오염을 나타냅니다. (F) 단핵구 정량 중에 피해야 큰 응집체를 형성 할 수있다. 볼 필드 무작위 선택을 사용하여, 패널 A는 MMA 제대로 수행되었는지 보통 관찰되는 것이다. 스케일 바는 20 μm의입니다. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55039/55039fig3large.jpg" target="_blank">여기를 클릭하세요</a>이 그림의 더 큰 버전을 볼 수 있습니다.

Watch this Scientific Journal Video about Use of a Monocyte Monolayer Assay to Evaluate FcÃŽÂ³ Receptor-mediated Phagocytosis at JoVE.com

Use of a Monocyte Monolayer Assay to Evaluate Fc&#947; Receptor-mediated Phagocytosis

The monocyte monolayer assay (MMA) is an in vitro assay that utilizes isolated primary monocytes obtained from mammalian peripheral whole blood to evaluate Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis.

단핵구 단층 분석의 사용은 Fcγ 수용체 - 매개 식균 작용을 평가하기

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly ...

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Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

17.8K 조회수.  University of Toronto. The monocyte monolayer assay (MMA) is an in vitro assay that utilizes isolated primary monocytes obtained from mammalian peripheral whole blood to evaluate Fcγ receptor (FcγR)-mediated phagocytosis.

비디오: Use of a Monocyte Monolayer Assay to Evaluate Fcγ Receptor-mediated Phagocytosis

Application of a Mouse Ligated Peyer&#x2019;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed to them and occasionally to pathogens. The gut-associated lymphoid tissue (GALT) play a key role for induction of the mucosal immune response to these microbes1, 2. To initiate the mucosal immune response, the mucosal antigens must be transported from the gut lumen across the epithelial barrier into organized lymphoid follicles such as Peyer's patches. This antigen transcytosis is mediated by specialized epithelial M cells3, 4. M cells are atypical epithelial cells that actively phagocytose macromolecules and microbes. Unlike dendritic cells (DCs) and macrophages, which target antigens to lysosomes for degradation, M cells mainly transcytose the internalized antigens. This vigorous macromolecular transcytosis through M cells delivers antigen to the underlying organized lymphoid follicles and is believed to be essential for initiating antigen-specific mucosal immune responses. However, the molecular mechanisms promoting this antigen uptake by M cells are largely unknown. We have previously reported that glycoprotein 2 (Gp2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane 5. Here, we present a method for the application of a mouse Peyer's patch intestinal loop assay to evaluate bacterial uptake by M cells. This method is an improved version of the mouse intestinal loop assay previously described 6, 7. The improved points are as follows: 1. Isoflurane was used as an anesthetic agent. 2. Approximately 1 cm ligated intestinal loop including Peyer's patch was set up. 3. Bacteria taken up by M cells were fluorescently labeled by fluorescence labeling reagent or by overexpressing fluorescent protein such as green fluorescent protein (GFP). 4. M cells in the follicle-associated epithelium covering Peyer's patch were detected by whole-mount immunostainig with anti Gp2 antibody. 5. Fluorescent bacterial transcytosis by M cells were observed by confocal microscopic analysis. The mouse Peyer's patch intestinal loop assay could supply the answer what kind of commensal or pathogenic bacteria transcytosed by M cells, and may lead us to understand the molecular mechanism of how to stimulate mucosal immune system through M cells.

Application of a Mouse Ligated Peyer&#8217;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

M cells in a specialized follicle-associated epithelium covering Peyer&#x2019;s patches play an important role for the mucosal immunosurveillance in gut-associated lymphoid tissue. Here we described the evaluation method for bacterial transcytosis by M cells in vivo. This method provides a method to understand M-cell function in the immune system.

마우스의 응용은 M 세포에 의해 박테리아 이해를 평가하는 Peyer의 패치 장 루프 분석 출혈도 잡았

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed ...

연구

면역학 및 감염병학

Use of Interferon-&gamma; Enzyme-linked Immunospot Assay to Characterize Novel T-cell Epitopes of Human Papillomavirus

A protocol has been developed to overcome the difficulties of isolating and characterizing rare T cells specific for pathogens, such as human papillomavirus (HPV), that cause localized infections. The steps involved are identifying region(s) of HPV proteins that contain T-cell epitope(s) from a subject, selecting for the peptide-specific T cells based on interferon-&gamma; (IFN-&gamma;) secretion, and growing and characterizing the T-cell clones (Fig. 1). Subject 1 was a patient who was recently diagnosed with a high-grade squamous intraepithelial lesion by biopsy and underwent loop electrical excision procedure for treatment on the day the T cells were collected1. A region within the human papillomavirus type 16 (HPV 16) E6 and E7 proteins which contained a T-cell epitope was identified using an IFN- g enzyme-linked immunospot (ELISPOT) assay performed with overlapping synthetic peptides (Fig. 2). The data from this assay were used not only to identify a region containing a T-cell epitope, but also to estimate the number of epitope specific T cells and to isolate them on the basis of IFN- &gamma; secretion using commercially available magnetic beads (CD8 T-cell isolation kit, Miltenyi Biotec, Auburn CA). The selected IFN-&gamma; secreting T cells were diluted and grown singly in the presence of an irradiated feeder cell mixture in order to support the growth of a single T-cell per well. These T-cell clones were screened using an IFN- &gamma; ELISPOT assay in the presence of peptides covering the identified region and autologous Epstein-Barr virus transformed B-lymphoblastoid cells (LCLs, obtained how described by Walls and Crawford)2 in order to minimize the number of T-cell clone cells needed. Instead of using 1 x 105 cells per well typically used in ELISPOT assays1,3, 1,000 T-cell clone cells in the presence of 1 x 105 autologous LCLs were used, dramatically reducing the number of T-cell clone cells needed. The autologous LCLs served not only to present peptide antigens to the T-cell clone cells, but also to keep a high cell density in the wells allowing the epitope-specific T-cell clone cells to secrete IFN-&gamma;. This assures successful performance of IFN-&gamma; ELISPOT assay. Similarly, IFN- &gamma; ELISPOT assays were utilized to characterize the minimal and optimal amino acid sequence of the CD8 T-cell epitope (HPV 16 E6 52-61 FAFRDLCIVY) and its HLA class I restriction element (B58). The IFN- &gamma; ELISPOT assay was also performed using autologous LCLs infected with vaccinia virus expressing HPV 16 E6 or E7 protein. The result demonstrated that the E6 T-cell epitope was endogenously processed. The cross-recognition of homologous T-cell epitope of other high-risk HPV types was shown. This method can also be used to describe CD4 T-cell epitopes4.

Use of Interferon-&#947; Enzyme-linked Immunospot Assay to Characterize Novel T-cell Epitopes of Human Papillomavirus

Characterizing T-cell epitopes of pathogens that cause localized infections such as human papillomavirus is a challenge because of limited number of T cells in circulation. A method is described in which rare T cells were isolated and were characterized starting with a very small number of cells.

인간 Papillomavirus의 소설 T-세포 Epitopes를 특성화하기 위해 인터페론-γ 효소 연결된 Immunospot 분석의 이용

A protocol has been developed to overcome the difficulties of isolating and characterizing rare T cells specific for pathogens, such as human ...

A Microplate Assay to Assess Chemical Effects on RBL-2H3 Mast Cell Degranulation: Effects of Triclosan without Use of an Organic Solvent

Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g. by an allergen), they degranulate, a process that results in the exocytosis of allergic mediators. Modulation of mast cell degranulation by drugs and toxicants may have positive or adverse effects on human health. Mast cell function has been dissected in detail with the use of rat basophilic leukemia mast cells (RBL-2H3), a widely accepted model of human mucosal mast cells3-5. Mast cell granule component and the allergic mediator &#946;-hexosaminidase, which is released linearly in tandem with histamine from mast cells6, can easily and reliably be measured through reaction with a fluorogenic substrate, yielding measurable fluorescence intensity in a microplate assay that is amenable to high-throughput studies1. Originally published by Naal et al.1, we have adapted this degranulation assay for the screening of drugs and toxicants and demonstrate its use here.
Triclosan is a broad-spectrum antibacterial agent that is present in many consumer products and has been found to be a therapeutic aid in human allergic skin disease7-11, although the mechanism for this effect is unknown. Here we demonstrate an assay for the effect of triclosan on mast cell degranulation. We recently showed that triclosan strongly affects mast cell function2. In an effort to avoid use of an organic solvent, triclosan is dissolved directly into aqueous buffer with heat and stirring, and resultant concentration is confirmed using UV-Vis spectrophotometry (using &#949;280 = 4,200 L/M/cm)12. This protocol has the potential to be used with a variety of chemicals to determine their effects on mast cell degranulation, and more broadly, their allergic potential.

Mast cell degranulation, the release of allergic mediators, is important in allergy, asthma, and parasite defense. Here we demonstrate techniques1 for assessing effects of drugs and toxicants on degranulation, methodology recently utilized to exhibit the powerful inhibitory effect of antibacterial agent triclosan2.

유기 용매를 사용하지 않고 트리클로산의 효과 : RBL-2H3 비만 세포 탈과립에 화학 효과를 평가하는 마이크로 분석 실험

Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g. by an allergen), they degranulate, a ...

Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro

Granulocytes play a key role in the body&#8217;s innate immune response to bacterial and viral infections. While methods exist to measure granulocyte function, in general these are limited in terms of the information they can provide. For example, most existing assays merely provide a percentage of how many granulocytes are activated following a single, fixed length incubation. Complicating matters, most assays focus on only one aspect of function due to limitations in detection technology. This report demonstrates a technique for simultaneous measurement of granulocyte phagocytosis of bacteria and oxidative burst. By measuring both of these functions at the same time, three unique phenotypes of activated granulocytes were identified: 1) Low Activation (minimal phagocytosis, no oxidative burst), 2) Moderate Activation (moderate phagocytosis, some oxidative burst, but no co-localization of the two functional events), and 3) High Activation (high phagocytosis, high oxidative burst, co-localization of phagocytosis and oxidative burst). A fourth population that consisted of inactivated granulocytes was also identified. Using assay incubations of 10, 20, and 40-min the effect of assay incubation duration on the redistribution of activated granulocyte phenotypes was assessed. A fourth incubation was completed on ice as a control. By using serial time incubations, the assay may be able to able to detect how a treatment spatially affects granulocyte function. All samples were measured using an image-based flow cytometer equipped with a quantitative imaging (QI) option, autosampler, and multiple lasers (488, 642, and 785 nm).

Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro

This method demonstrates a technique for assessing granulocyte function by simultaneously measuring phagocytosis of bacteria and oxidative burst. Image-based flow cytometry allowed for the identification of three distinct subsets of activated granulocytes that all differed in their relative functional capacity.

이미지 기반 유동 세포 계측법 기술은 과립구 기능의 변화를 평가하기<em&gt; 체외</em

Granulocytes play a key role in the body&#8217;s innate immune response to bacterial and viral infections. While methods exist to measure granulocyte ...

Measuring Granulocyte and Monocyte Phagocytosis and Oxidative Burst Activity in Human Blood

The granulocyte and monocyte phagocytosis and oxidative burst (OB) activity assay can be used to study the innate immune system. This manuscript provides the necessary methodology to add this assay to an exercise immunology arsenal. The first step in this assay is to prepare two aliquots ("H" and "F") of whole blood (heparin). Then, dihydroethidium is added to the H aliquot, and both aliquots are incubated in a warm water bath followed by a cold water bath. Next, Staphylococcus aureus (S. aureus) is added to the H aliquot and fluorescein isothiocyanate-labeled S. aureus is added to the F aliquot (bacteria:phagocyte = 8:1), and both aliquots are incubated in a warm water bath followed by a cold water bath. Then, trypan blue is added to each aliquot to quench extracellular fluorescence, and the cells are washed with phosphate-buffered saline. Next, the red blood cells are lysed, and the white blood cells are fixed. Finally, a flow cytometer and appropriate analysis software are used to measure granulocyte and monocyte phagocytosis and OB activity. This assay has been used for over 20 years. After heavy and prolonged exertion, athletes experience a significant but transient increase in phagocytosis and an extended decrease in OB activity. The post-exercise increase in phagocytosis is correlated with inflammation. In contrast to normal weight individuals, granulocyte and monocyte phagocytosis is chronically elevated in overweight and obese participants, and is modestly correlated with C-reactive protein. In summary, this flow cytometry-based assay measures the phagocytosis and OB activity of phagocytes and can be used as an additional measure of exercise- and obesity-induced inflammation.

The purpose of this manuscript is to present a method for measuring monocyte and granulocyte phagocytosis and oxidative burst activity in human blood samples.

인간의 혈액에서 측정 과립구와 단핵구 식균 작용 및 산화 버스트 활동

The granulocyte and monocyte phagocytosis and oxidative burst (OB) activity assay can be used to study the innate immune system. This manuscript provides ...

Characterization of Human Monocyte-derived Dendritic Cells by Imaging Flow Cytometry: A Comparison between Two Monocyte Isolation Protocols

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and pathogenesis of inflammation. Isolation and study of DCs have been important in biological research because of their distinctive features. Although they are essential key mediators of the immune system, DCs are very rare in blood, accounting for approximately 0.1 - 1% of total blood mononuclear cells. Therefore, alternatives for isolation methods rely on the differentiation of DCs from monocytes isolated from peripheral blood mononuclear cells (PBMCs). The utilization of proper isolation techniques that combine simplicity, affordability, high purity, and high yield of cells is imperative to consider. In the current study, two distinct methods for the generation of DCs will be compared. Monocytes were selected by adherence or negatively enriched using magnetic separation procedure followed by differentiation into DCs with IL-4 and GM-CSF. Monocyte and MDDC viability, proliferation, and phenotype were assessed using viability dyes, MTT assay, and CD11c/ CD14 surface marker analysis by imaging flow cytometry. Although the magnetic separation method yielded a significant higher percentage of monocytes with higher proliferative capacity when compared to the adhesion method, the findings have demonstrated the ability of both techniques to simultaneously generate monocytes that are capable of proliferating and differentiating into viable CD11c+ MDDCs after seven days in culture. Both methods yielded &gt; 70% CD11c+ MDDCs. Therefore, our results provide insights that contribute to the development of reliable methods for isolation and characterization of human DCs.

This study compares two different methods of human monocyte isolation for obtaining in vitro dendritic cells (DCs). Monocytes are selected by adherence or negatively enriched by magnetic separation. Monocyte yield and viability along with MDDC viability, proliferation and CD11c/CD14 surface marker expression will be compared between both methods.

이미징 유동 세포 계측법에 의해 인간 단핵구 유래 수지상 세포의 특성 : 두 단핵구 격리 프로토콜 사이의 비교

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and ...

Use of the Soft-agar Overlay Technique to Screen for Bacterially Produced Inhibitory Compounds

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, including work with bacteriophages and bacteriocins, proteinaceous antibacterial agents. This approach is relatively inexpensive, with minimal resource requirements. This technique consists of spotting supernatant from a donor strain (potentially harboring a toxic compound(s)) onto a solidified soft agar overlay that is seeded with a bacterial test strain (potentially sensitive to the toxic compound(s)). We utilized this technique to screen a library of Pseudomonas syringae strains for intraspecific killing. By combining this approach with a precipitation step and targeted gene deletions, multiple toxic compounds produced by the same strain can be differentiated. The two antagonistic agents commonly recovered using this technique are bacteriophages and bacteriocins. These two agents can be differentiated using two simple additional tests. Performing a serial dilution on a supernatant containing bacteriophage will result in individual plaques becoming less in number with greater dilution, whereas serial dilution of a supernatant containing bacteriocin will result a clearing zone that becomes uniformly more turbid with greater dilution. Additionally, a bacteriophage will produce a clearing zone when spotted onto a fresh soft agar overlay seeded with the same strain, whereas a bacteriocin will not produce a clearing zone when transferred to a fresh soft agar lawn, owing to the dilution of the bacteriocin.

We describe a simple method for screening bacterial cultures for the production of compounds inhibitory towards other bacteria.

소프트 한천 오버레이 기법의 사용은 세균 생산 억제 화합물에 대한 화면으로

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, ...

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to effectiveness in the clinical setting. This is likely due to a lack of consideration for the physiologically relevant cellular penetration barriers that exist in the infected host. We recently established an alternative infection model that generates large macrophage aggregate structures containing densely packed M. tuberculosis (Mtb) at its core, which was suitable for drug susceptibility testing. This infection model is inexpensive, rapid, and most importantly BSL-2 compatible. Here, we describe the experimental procedures to generate Mtb/macrophage aggregate structures that would produce macrophage-passaged Mtb for drug susceptibility testing. In particular, we demonstrate how this infection system could be directly adapted to the 96-well plate format showing throughput capability for the screening of compound libraries against Mtb. Overall, this assay is a valuable addition to the currently available Mtb drug discovery toolbox due to its simplicity, cost effectiveness, and scalability.

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

New models and assays that would improve the early drug development process for next-generation anti-tuberculosis drugs are highly desirable. Here, we describe a quick, inexpensive, and BSL-2 compatible assay to evaluate drug efficacy against Mycobacterium tuberculosis that can be easily adapted for high-throughput screening.

높은 처리량을 지원 분석은 대식 세포 계대에 대한 약물 효능을 평가하기<em&gt; 결핵균</em

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to ...

Phagocytosis Assay for Apoptotic Cells in Drosophila Embryos

The molecular mechanisms underlying the phagocytosis of apoptotic cells need to be elucidated in more detail because of its role in immune and inflammatory intractable diseases. We herein developed an experimental method to investigate phagocytosis quantitatively using the fruit fly Drosophila, in which the gene network controlling engulfment reactions is evolutionally conserved from mammals. In order to accurately detect and count engulfing and un-engulfing phagocytes using whole animals, Drosophila embryos were homogenized to obtain dispersed cells including phagocytes and apoptotic cells. The use of dispersed embryonic cells enables us to measure in vivo phagocytosis levels as if we performed an in vitro phagocytosis assay in which it is possible to observe all phagocytes and apoptotic cells in whole embryos and precisely quantify the level of phagocytosis. We confirmed that this method reproduces those of previous studies that identified the genes required for the phagocytosis of apoptotic cells. This method allows the engulfment of dead cells to be analyzed, and when combined with the powerful genetics of Drosophila, will reveal the complex phagocytic reactions comprised of the migration, recognition, engulfment, and degradation of apoptotic cells by phagocytes.

Phagocytosis Assay for Apoptotic Cells in Drosophila Embryos

We herein describe a phagocytosis assay using the dispersed embryonic cells of Drosophila. It enables us to easily and precisely quantify in vivo phagocytosis levels, and to identify new molecules required for the phagocytosis of apoptotic cells.

에있는 Apoptotic 세포를위한 Phagocytosis 검정<em&gt; Drosophila</em&gt; 태아

The molecular mechanisms underlying the phagocytosis of apoptotic cells need to be elucidated in more detail because of its role in immune and ...

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

The aim of this work is to show a novel method to evaluate the ability of some immunomodulatory molecules, such as antimicrobial peptides (AMPs), to stimulate cell migration. Importantly, cell migration is a rate-limiting event during the wound-healing process to re-establish the integrity and normal function of tissue layers after injury. The advantage of this method over the classical assay, which is based on a manually made scratch in a cell monolayer, is the usage of special silicone culture inserts providing two compartments to create a cell-free pseudo-wound field with a well-defined width (500 &#956;m). In addition, due to an automated image analysis platform, it is possible to rapidly obtain quantitative data on the speed of wound closure and cell migration. More precisely, the effect of two frog-skin AMPs on the migration of bronchial epithelial cells will be shown. Furthermore, pretreatment of these cells with specific inhibitors will provide information on the molecular mechanisms underlying such events.

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

Here, we present a protocol to evaluate the effect of peptides on the migration of bronchial epithelial cells. This method allows for the rapid and highly reproducible obtainment of quantitative data on the speed of cell migration and wound closure.

상처 치유 분석 결과 셀 마이그레이션 평가 하는 새로운 체 외에

The aim of this work is to show a novel method to evaluate the ability of some immunomodulatory molecules, such as antimicrobial peptides (AMPs), to ...