MRS을 부여하여 부분 지원 자연 과학, 캐나다 공학 연구 협의회 (NSERC), 신경 과학 연구, ARC에 대한 해리 Botterell 재단, 그리고 가필드 켈리 순환기 연구 및 개발 기금에서입니다. SRA는 여왕의 대학 연​​구 대상과 로버트 J. 윌슨의 원정받는 사람입니다. PJ는 여왕의 졸업 수상과 로버트 J. 윌슨의 원정받는 사람입니다. AG와 SA는 여왕의 대학원 수상 수상자입니다. SF는 과학 기술 (OGSST)에서 온타리오 대학원 장학금받는했다. AG는 여왕의 프랭클린 브래켄 대학원 장학금받는 것입니다. SA는 여왕의 RS 맥롤린 대학원 장학금받는 것입니다.

1. 냉동 세포 대식 세포의 세포를 부활 <ol><li> -80에서 냉동 세포를 부활하기 전에 ° C 냉장고 10 % 태아 소 혈청 (FBS) 및 5 μg / plasmocin의 ML과 보충 살균 필터 Dulbecco의 수정된 이글의 중간 (DMEM)을 사용하여 배지를 준비 하나 필요합니다. Plasmocin은 세포의 문화 mycoplasma 오염을 제거하고 방지하기 위해 우리의 연구에 사용되는 항생제 솔루션입니다. </li><li> 냉동 세포 중 하나가 병에 들어, 4 ML, 살균 생물 학적 봉쇄 후드에서 25cm 2 세포 배양 플라스크에 교양 매체 1 ML을 추가합니다. </li><li> 냉동 세포의 유리병은 -80 ° C의 냉동고에서 가져온 경우, 그들은 빠르게 약 2-3 분 소요 멸균 후드에 손 온난화에 의해 해동됩니다. </li><li> 즉시 세포가 해동되면서, 그들은 신속하게 1mL 피펫을 사용하여 살균 시설이 매체를 포함하고있는 술병으로 전송됩니다. </li><li> 세포를 포함하는 플라스크는 37 ° C와 5% CO 2에서 조직 문화 인큐베이터에 배치됩니다. </li><li> 그들이 거꾸로 현미경을 사용하여 75 % 90% 합류가 될 때까지 세포는 매일 성장과 준수를위한 검사. </li><li> 세포 배양 보육 동안, 12mm 원형 유리 슬라이드는 생물 학적 봉쇄 캐비닛에 1 시간을 위해 큰 유리는 배양 접시에 70 % 에탄올에서 그들을 다루는 소독 있으며, 알코올은 나중에 제거 reusage 보관하고, 젖은 유리 측면입니다 24 시간 또는 그들이 건조 때까지 UV 아래 캐비닛에 멸균 필터링 공기 흐름에 노출 남아 있습니다. </li></ol> 2. Sialidase 분석을위한 도금 셀 <ol><li> 세포를 도금하기 전에 하나의 무균 원형 유리 슬라이드는 신중하게 스테인레스 스틸 forcep, 톱니 모양의, &quot;4, 소독 불타는 전체 곡선을 사용하여 BD 팰컨 잘 하나 24 잘 뚜껑이있는 평면 바닥 조직 문화 처리 폴리스티렌 판 배치됩니다 . 75 %에 합류 세포 중 하나가 술병을 위해, 우리는 대개 문화 판 12 우물을 사용합니다. </li><li> 자기편 대식 세포 세포 들어, DMEM 시설 매체는 멸균 용기 캐비닛에 5 ML 멸균 피펫을 사용하여 플라스크에서 제거됩니다. 1X 무균 트리스와 함께 한번 자기편 세포를 씻고있는 혈청이 포함된 미디어를 제거하고 세포를 감추기에 충분 할만큼 산도 7.4에서 호수 버퍼 칼슘 매체 무료 (CMF) 인산 1 ML를 추가하는 생리 산도 7.4이 버퍼. 세포는 술병에서 발사 전까지는 세포는 약 10 분 또는 37 ° C 배양기에 배치됩니다. 플라스크는 부드럽게 누볐어이나 남아있는 자기편 세포를 풀어야 흔들리고 수 있습니다. </li><li> 술병에서 중단 세포의 제 1 ML하려면, DMEM 시설 중간 3 ML를 추가합니다. 정지 세포의 0.5 ML를 타고 sterilely 12mm 원형 유리 슬라이드를 포함하는 조직 교양 판에게 양도. 약 0.5 X 10 6 세포는 원형 유리 슬라이드로 전송됩니다. </li><li> 조직 배양 플레이트 포함하는 세포는 37 incubated 아르 ° C 5 % 세포가 원형 유리 슬라이드에 충실하기 위해 수 있도록 24 시간에 대한 CO 2. </li></ol> 3. Sialidase 분석 1. 컨트롤 만들기 <ol><li> 봉쇄 캐비닛 또는 실험실 벤치에서 순서대로 다음과 같은 성분 혼합하여 추가 : (1 엔드, 1slide, 25 X 75 X 1mm 프로스트) : 현미경 슬라이드를 삽입 4 MUNANA 1 μL DAKO + 1 μL를 끝으로 + 트리스 3 μL는 생리 산도 7.4 (TBS)을 버퍼. </li><li> 잘 세포를 포함하는 하나의 미디어를 제거한 후, 부드럽게 잘의 모퉁이에 4 &quot;forcep으로 원형 유리 슬라이드 리프트. 조심스럽게 forcep으로 유리 슬라이드를 제거 및 혼합 솔루션에 직면하고있는 세포로 슬라이드를 장소 현미경 슬라이드. </li><li> 바로, UV 필터 설정하고 형광 이미지를 캡처를위한 디지털 카메라를 가지고있다 에피 형광 현미경으로 microcope 슬라이드 가져가라. </li><li> 당신은 형광등 모드로 전환 후 세포를 볼 수 있고 때까지 위상 대조에서 현미경을 집중. 1 분, 2 분, 3 분 형광등 아래에있는 세포의 사진을 가져가라. 위상 대조 아래 세포의 단일 사진을 가져가라. </li></ol> 2. 긍정적인 테스트 만들기 <ol><li> 4 MUNANA 1 μL DAKO + 1 μL LPS의 + 2 μL + TBS 1 μL : 다른 현미경 슬라이드에있는 순서대로 다음과 같은 화합물 스팟. </li><li> 위의 단계 3.1.2에 3.1.4를 반복합니다. </li></ol> 3. Neuraminidase 억제제는 Tamiflu와 함께 긍정적인 테스트 만들기 <ol><li> 다른 현미경 슬라이드에 다음과 같은 순서에 다음과 같은 화합물을 스팟 : 4 MUNANA의 1μL DAKO + 1μL + LPS 2 μL + Tamiflu 1 μL. </li><li> 원형 유리 슬라이드에 살고 세포와 접촉하는 물질의 최종 농도가 5 희석 계수를해야합니다, Tamiflu의 농도는 생리 산도 7.4 버퍼 1X 트리스의 사전 준비. </li><li> 위의 단계 3.1.2에 3.1.4를 반복합니다. </li></ol> 4. 억제제의 농도의 결정은 Sialidase 활동의 50 % (IC 50) 억제하는 데 필요한 <ol><li> 세포를 둘러싼 형광을 계​​량하기 위해 이미지는 RBG을 측정하는 분석 플러그인과 ImageJ 1.38x를 사용하여 분석하고 있습니다. 세포를 둘러싸고있는 형광 50 무작위 현장 판독을 가지고 총 형광의 평균을 계산합니다. </li><li> IC 50 비선형 회귀를 사용하여 Y 축의 평균 형광으로부터 X 축에 (예를 들어, 로그 NG / ML) 로그 단위로 변환 억제제 농도의 플롯에서 생성됩니다. </li></ol> 5. 성공의 비밀 <ol><li> 문화의 세포가 mycoplasma에서 무료로 오염되어 있는지 확인하십시오. 우리는 일상적으로 이에 대한 제어하는​​ 문화 매체 Plasmocin을 사용합니다. </li><li> 인공 neuraminidase 기판, 4 MUNANA는 신선한 사용해야합니다. 준비 기판은 sialidase 분석을위한 일주일 이내 사용할 수 있습니다. </li><li> 세포주의 세포 표면에 낮은 수용체 표현이있다면, 하나는 자극에 대한 리간드의 높은 복용량을 사용할 수 있습니다. </li><li> 우리는 또한 이상의 여섯 번 passaged 세포들의 수용체 표현형을 잃을 수도 경험이있다. 갓, 부활 전지 교양하여야한다. </li></ol> 6. 대표 결과 첨부된을 대표하는 결과 sialidase 분석의 애니메이션 프로토콜 참조 <a href="http://www.jove.com/files/ftp_upload/2142/szewczuk_JoVE_sialidase_assay.pptx">PowerPoint 파일을</a> .

<ol>
	<li>Amith, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neu1+desialylation+of+sialyl+alpha-2,3-linked+beta-galactosyl+residues+of+TOLL-like+receptor+4+is+essential+for+receptor+activation+and+cellular+signaling.">Neu1 desialylation of sialyl alpha-2,3-linked beta-galactosyl residues of TOLL-like receptor 4 is essential for receptor activation and cellular signaling.</a> Cell Signal. 22, 314-324 (2010).</li><li>Amith, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dependence+of+pathogen+molecule-induced+Toll-like+receptor+activation+and+cell+function+on+Neu1+sialidase.">Dependence of pathogen molecule-induced Toll-like receptor activation and cell function on Neu1 sialidase.</a> Glycoconjugate Journal. 26, 1197-1212 (2009).</li><li>Woronowicz, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dependence+of+neurotrophic+factor+activation+of+Trk+tyrosine+kinase+receptors+on+cellular+sialidase.">Dependence of neurotrophic factor activation of Trk tyrosine kinase receptors on cellular sialidase.</a> Glycobiology. 17, 10-24 (2007).</li><li>Jayanth, P., Amith, S. R., Gee, K., Szewczuk, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neu1+Sialidase+and+Matrix+Metalloproteinase-9+Cross-talk+is+Essential+for+Neurotrophin+Activation+of+Trk+Receptors+and+Cellular+Signaling.">Neu1 Sialidase and Matrix Metalloproteinase-9 Cross-talk is Essential for Neurotrophin Activation of Trk Receptors and Cellular Signaling.</a> Cell. Signal. 22, 1193-1205 (2010).</li><li>Finlay, T. M., Jayanth, P., Amith, S. R., Glimour, A., Guzzo, C., Gee, K., Beyaert, R., Szewczuk, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thymoquinone+from+nutraceutical+black+cumin+oil+activates+Neu4+sialidase+in+live+macrophage,+dendritic,+and+normal+and+type+I+sialidosis+human+fibroblast+cells+via+GPCR+G&#945;i+proteins+and+matrix+metalloproteinase-9.">Thymoquinone from nutraceutical black cumin oil activates Neu4 sialidase in live macrophage, dendritic, and normal and type I sialidosis human fibroblast cells via GPCR G&#945;i proteins and matrix metalloproteinase-9.</a> Glycoconj J. 27, 329-348 (2010).</li><li>Seyrantepe, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+Phagocytosis+in+Macrophages+by+Neuraminidase+1.">Regulation of Phagocytosis in Macrophages by Neuraminidase 1.</a> Journal of Biological Chemistry. 285, 206-215 (2010).</li></ol>

SRA와 PJ 똑같이 첫번째 저자로 기여.

라이브 대식 세포 세포 2 sialidase 활동을 감지하기 위해 새로 개발된 분석을 사용하여, 우리는 BMC - 2 복용량에 의존 방식으로뿐만 아니라 사는 대식 세포의 세포 DC - 2.4 돌기 세포에 거주 리간드 유도된 sialidase 활동에 sialidase 활동을 감지하는이 기술을 사용 , HEK-TLR4/MD2, HEK293, SP1 내유 선암 세포, 인간 WT 및 1140F01 및 WG0544 종류 I sialidosis의 fibroblast 세포. Tamiflu (oseltamivir 인산염)과 같은 Neuramini...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>DMEM</td>
<td>&nbsp;</td>
<td>Gibco, Rockville, MD</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>4-MUNANA</td>
<td>&nbsp;</td>
<td>Biosynth International Inc., Itasca, IL, USA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Fetal calf serum</td>
<td>&nbsp;</td>
<td>HyClone, Logan, Utah, USA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>DakoCytomation, Fluorescent Mounting Media</td>
<td>&nbsp;</td>
<td>DAKO North America, Inc., Carpintenia, CA</td>
<td>S3023</td>
<td>15 mL</td>
</tr>
<tr>
<td>Tamiflu (Oseltamivir Phosphate)</td>
<td>&nbsp;</td>
<td>Hoffman La Roche</td>
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detection of  neu1 sialidase activity in regulating toll-like receptor activation

포유류의 수신자 같은 수용체 (TLRs)는 병원균 - 관련 분자 패턴을 인식 수용체의 가족입니다. 뿐만 아니라 TLRs는 미생물의 중요한 센서 (예, 바이러스, 세균 및 기생충) 감염되며, 그들은 또한 가능성이 면역 질환으로 전염성 질환, 염증 질환, 그리고 pathophysiology에 중요한 역할을한다. 따라서, 감염증에 대한 TLR의 반응의 강도와 기간은 강력히 제어해야합니다. 이 센서 수용체들은 리간드 상호 작용과 신호 구성 요소의 구조적 무결성을 이해하는 것은 이후의 면역 보호를 위해 필수적인 것이 다음과 같습니다. 또한 센서 조작을 통해 질병 조작을위한 중요한 기회를 제공합니다. TLR 센서의 신호 경로가 잘 묘사되고 있지만, 매개 변수는 센서와 그 리간드 여전히 저조한 정의 남아 사이의 상호 작용을 통제. 우리는 최근 이전에 1,2을 관찰되지 않은 자연 리간드에 의해 TLR 활성화의 소설 메커니즘을 확인했습니다. 그것은 리간드 유도된 TLR 활성화가 밀접하게 Neu1 sialidase 활성화에 의해 제어됩니다 것을 제안합니다. 우리는 또한 Neu1가 단단히 Neu1 및 매트릭스 metalloproteinase - 9 (MMP - 9) 수용체 4 단지에 간 이야기를 포함 TrkA와 TrkB 3 번 neurotrophin 수용체를 조절보고있다. sialidase 분석 처음 GPCR Gαi 단백질 및 MMP - 9 5를 통해 macrophages, 돌기 세포 fibroblast 세포의 세포 표면에 Neu4 sialidase의 활성화에 새로운 리간드, thymoquinone를 찾을 사용되었습니다. TLR 수용체에 대한 우리의 데이터는 Neu1 sialidase는 TLR - 2, -3 및 -4 수용체 복잡한 이미이며, 두 수용체에 리간드 바인딩에 유도되고있는 것으로 나타났습니다. 활성 Neu1 sialidase의 hydrolyzes sialyl α - 2 TLR - 4 dimerization, MyD88/TLR4 복잡한 채용, NFkB의 활성화와 프로 염증성 세포 반응에 steric hinderance를 제거하는 바인딩 리간드에서 멀리 ,3 - 링크 β - galactosyl 잔류물. 공동 보고서에서 Neu1의 sialidase는 대식 세포 세포 6 phagocytosis 조절을 보여줘왔다. 함께 촬영 sialidase 분석은 리간드 유도된 수용체 활성화의 분자 메커니즘에 강력한 통찰력으로 우리를 제공합니다. Neu1 sialidase과 TLR의 활성화 사이의 정확한 관계가, Trk 수용체는 아직 완전히 elucidated 수 있지만, 그것은 세포 규제 경로에 신규 또는 선구적인 접근 방식을 나타내는 것입니다.

Watch this Scientific Journal Video about Detection of  Neu1 Sialidase Activity in Regulating TOLL-like Receptor Activation at JoVE.com

Detection of  Neu1 Sialidase Activity in Regulating TOLL-like Receptor Activation

sialidase 분석은 미생물의 감염 라이브 macrophages의 세포 표면에 수용체 수준에서 염증성 질환에 관여 TLR 센서 소설 분자 메커니즘 (들)을 명료하게하다하는 간단한 기술 접근​​이다.

수신자 같은 수용체 활성화 레굴라팅에 Neu1 Sialidase 활동 감지

Mammalian Toll-like receptors (TLRs) are a family of receptors that recognize pathogen-associated molecular patterns. Not only are TLRs crucial sensors of ...

detection-neu1-sialidase-activity-regulating-toll-like-receptor

Research

JoVE Journal

Immunology and Infection

11.5K 조회수.  Queen's University - Kingston, Ontario. sialidase 분석은 미생물의 감염 라이브 macrophages의 세포 표면에 수용체 수준에서 염증성 질환에 관여 TLR 센서 소설 분자 메커니즘 (들)을 명료하게하다하는 간단한 기술 접근​​이다.

비디오: Detection of  Neu1 Sialidase Activity in Regulating TOLL-like Receptor Activation

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

접착제 테이프 기반의 샘플링 및 형광의 조합 현장에서</em의 신속한 감지를 위해> 하이브 리다이 제이션 살모넬라균 신선한 농산물에 대한

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

연구

면역학 및 감염병학

Basophil Activation Test for Investigation of IgE-Mediated Mechanisms in Drug Hypersensitivity

Hypersensitivity reactions against non-steroidal anti-inflammatory drugs (NSAIDs) like propyphenazone (PP) and diclofenac (DF) can manifest as Type I-like allergic reactions 1. In clinical practice, diagnosis of drug hypersensitivity is mainly performed by patient history, as skin testing is not reliable and oral provocation testing bears life-threatening risks for the patient 2. Hence, evidence for an underlying IgE-mediated pathomechanism is hard to obtain. 
Here, we present an in vitro method based on the use of human basophils derived from drug-hypersensitive patients that mimics the allergic effector reaction in vivo. As basophils of drug-allergic patients carry IgE molecules specific for the culprit drug, they become activated upon IgE receptor crosslinking and release allergic effector molecules. The activation of basophils can be monitored by the determination of the upregulation of CD63 surface expression using flow cytometry 3. 
In the case of low molecular weight drugs, conjugates are designed to enable IgE receptor crosslinking on basophils. As depicted in Figure 1, two representatives of NSAIDs, PP and DF, are covalently bound to human serum albumin (HSA) via a carboxyl group reacting with the primary amino group of lysine residues. DF carries an intrinsic carboxyl group and, thus, can be used directly 4, whereas a carboxyl group-containing derivative of PP had to be organochemically synthesized prior to the study 1.
The coupling degree of the low molecular weight compounds on the protein carrier molecule and their spatial distribution is important to guarantee crosslinking of two IgE receptor molecules. The here described protocol applies high performance-size exclusion chromatography (HPSEC) equipped with a sequential refractive index (RI) and ultra violet (UV) detection system for determination of the coupling degree.
As the described methodology may be applied for other drugs, the basophil activation test (BAT) bears the potential to be used for the determination of IgE-mediated mechanisms in drug hypersensitivity. Here, we determine PP hypersensitivity as IgE-mediated and DF hypersensitivity as non-IgE-mediated by BAT.

Basophil activation test is a potent tool for the detection of IgE-dependent allergies in vitro. Here, an optimized protocol for basophil activation test is used to investigate drug hypersensitivity. A method for the efficient production of covalent drug-protein conjugates and their physicochemical characterization is described.

약물 과민성의 IGE - 중재 메커니즘을 조사 Basophil 인증 시험

Hypersensitivity reactions against non-steroidal anti-inflammatory drugs (NSAIDs) like propyphenazone (PP) and diclofenac (DF) can manifest as Type I-like ...

Quantitative Imaging of Lineage-specific Toll-like Receptor-mediated Signaling in Monocytes and Dendritic Cells from Small Samples of Human Blood

Individual variations in immune status determine responses to infection and contribute to disease severity and outcome. Aging is associated with an increased susceptibility to viral and bacterial infections and decreased responsiveness to vaccines with a well-documented decline in humoral as well as cell-mediated immune responses1,2. We have recently assessed the effects of aging on Toll-like receptors (TLRs), key components of the innate immune system that detect microbial infection and trigger antimicrobial host defense responses3. In a large cohort of healthy human donors, we showed that peripheral blood monocytes from the elderly have decreased expression and function of certain TLRs4 and similar reduced TLR levels and signaling responses in dendritic cells (DCs), antigen-presenting cells that are pivotal in the linkage between innate and adaptive immunity5. We have shown dysregulation of TLR3 in macrophages and lower production of IFN by DCs from elderly donors in response to infection with West Nile virus6,7. 
Paramount to our understanding of immunosenescence and to therapeutic intervention is a detailed understanding of specific cell types responding and the mechanism(s) of signal transduction. Traditional studies of immune responses through imaging of primary cells and surveying cell markers by FACS or immunoblot have advanced our understanding significantly, however, these studies are generally limited technically by the small sample volume available from patients and the inability to conduct complex laboratory techniques on multiple human samples. ImageStream combines quantitative flow cytometry with simultaneous high-resolution digital imaging and thus facilitates investigation in multiple cell populations contemporaneously for an efficient capture of patient susceptibility. Here we demonstrate the use of ImageStream in DCs to assess TLR7/8 activation-mediated increases in phosphorylation and nuclear translocation of a key transcription factor, NF-&kappa;B, which initiates transcription of numerous genes that are critical for immune responses8. Using this technology, we have also recently demonstrated a previously unrecognized alteration of TLR5 signaling and the NF-&kappa;B pathway in monocytes from older donors that may contribute to altered immune responsiveness in aging9.

We describe use of ImageStream technology (<a href="http://www.amnis.com/" target="_blank">www.amnis.com</a>), which combines quantitative flow cytometry with simultaneous high-resolution digital imaging, to quantify cellular mechanisms of primary immune cells from well-defined patient cohorts. Our studies provide a blueprint for translational investigations to quantify lineage specific cellular responses in small samples from subject cohorts.

Monocytes과 인간 혈액의 작은 샘플에서 돌기 세포의 리니지 특정 수신자 같은 수용체 - 매개 시그널링의 정량 이미징

Individual variations in immune status determine responses to infection and contribute to disease severity and outcome.  Aging is associated with an ...

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

Ixodes Scapularis 진드기의 타액, 침샘 및 Hemolymph 컬렉션

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

Methods for Quantitative Detection of Antibody-induced Complement Activation on Red Blood Cells

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver (extravascular hemolysis) or leading to intravascular lysis of RBCs. Alloantibodies (e.g. ABO) or autoantibodies to RBC antigens (as seen in autoimmune hemolytic anemia, AIHA) leading to complement activation are potentially harmful and can be - especially when leading to intravascular lysis - fatal1. Currently, complement activation due to (auto)-antibodies on RBCs is assessed in vitro by using the Coombs test reflecting complement deposition on RBC or by a nonquantitative hemolytic assay reflecting RBC lysis1-4. However, to assess the efficacy of complement inhibitors, it is mandatory to have quantitative techniques. Here we describe two such techniques. First, an assay to detect C3 and C4 deposition on red blood cells that is induced by antibodies in patient serum is presented. For this, FACS analysis is used with fluorescently labeled anti-C3 or anti-C4 antibodies. Next, a quantitative hemolytic assay is described. In this assay, complement-mediated hemolysis induced by patient serum is measured making use of spectrophotometric&#160;detection of the released hemoglobin. Both of these assays are very reproducible and quantitative, facilitating studies of antibody-induced complement activation.

Here we describe two assays for measuring complement activation induced by antibodies against red blood cells. The major advantage over the current assays is their quantitative and easy-to-interpret nature.

적혈구에 대한 항체에 의한 보체 활성의 정량 검출하는 방법

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver ...

Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1&#946; in Human Monocyte-derived Dendritic Cells

Inflammatory processes resulting from the secretion of Interleukin (IL)-1 family cytokines by immune cells lead to local or systemic inflammation, tissue remodeling and repair, and virologic control1,2 . Interleukin-1&#946; is an essential element of the innate immune response and contributes to eliminate invading pathogens while preventing the establishment of persistent infection1-5.
Inflammasomes are the key signaling platform for the activation of interleukin 1 converting enzyme (ICE or Caspase-1). The NLRP3 inflammasome requires at least two signals in DCs to cause IL-1&#946; secretion6. Pro-IL-1&#946; protein expression is limited in resting cells; therefore a priming signal is required for IL-1&#946; transcription and protein expression. A second signal sensed by NLRP3 results in the formation of the multi-protein NLRP3 inflammasome. The ability of dendritic cells to respond to the signals required for IL-1&#946; secretion can be tested using a synthetic purine, R848, which is sensed by TLR8 in human monocyte derived dendritic cells&#160;(moDCs) to prime cells, followed by activation of the NLRP3 inflammasome with the bacterial toxin and potassium ionophore, nigericin.
Monocyte derived DCs&#160;are easily produced in culture and provide significantly more cells than purified human myeloid DCs. The method presented here differs from other inflammasome assays in that it uses in vitro human, instead of mouse derived, DCs thus allowing for the study of the inflammasome in human disease and infection.

Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1&amp;#946; in Human Monocyte-derived Dendritic Cells

Dendritic cells (DCs)&#160;secrete IL-1&#946; in response to TLR8 recognition of synthetic purine, R848, followed by NLRP3 inflammasome activation with nigericin, therefore, IL-1&#946; can be used to measure NLRP3 inflammasome activity. Intracellular cytokine staining, immunoblotting, and ELISA are used to accurately measure NLRP3 inflammasome priming and activation via IL-1&#946; expression.

활성화 및 인간 단핵구 유래 수지상 세포에서 IL-1β를 사용하여 NLRP3 inflammasome을 활동의 측정

Inflammatory processes resulting from the secretion of Interleukin (IL)-1 family cytokines by immune cells lead to local or systemic inflammation, tissue ...

Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. Macrophages express surface Toll-like receptors (TLRs), which recognize molecular patterns conserved through evolution in a wide range of microorganisms. TLRs play a central role in macrophage activation which is usually associated with gene expression alteration. Macrophages are critical in many diseases and have emerged as attractive targets for therapy. In the following protocol, we describe a procedure to isolate murine peritoneal macrophages using Brewer&#8217;s thioglycollate medium. The latter will boost monocyte migration into the peritoneum, accordingly this will raise macrophage yield by 10-fold. Several studies have been carried out using bone marrow, spleen or peritoneal derived macrophages. However, peritoneal macrophages were shown to be more mature upon isolation and are more stable in their functionality and phenotype. Thus, macrophages isolated from murine peritoneal cavity present an important cell population that can serve in different immunological and metabolic studies. Once isolated, macrophages were stimulated with different TLR ligands and consequently&#160;gene expression was evaluated.

We describe here a simple protocol to isolate murine peritoneal macrophages. This procedure is followed by RNA extraction to carry out gene expression analysis upon Toll-like receptors stimulation.

쥐 복막 대 식세포의 분리는 수신자 같은 수용체 자극하면 유전자 발현 분석을 수행하기

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. ...

Isolation and Activation of Murine Lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

분리 및 쥐 림프구의 활성화

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

인간의 전체 혈액 자연 살해 세포 용 균성 활동의 유세포 분석

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote inflammatory and autoinflammatory diseases. Inflammasomes are activated by cytosolic pattern recognition receptors, including members of the NOD-like receptor (NLR) family. These receptors oligomerize upon the detection of microbial or damage-associated stimuli. Subsequent recruitment of the adaptor protein ASC forms a microscopically visible inflammasome complex, which activates caspase-1 through proximity-induced auto-activation. Following the activation, caspase-1 cleaves pro-IL-1&#946; and pro-IL-18, leading to the activation and secretion of these pro-inflammatory cytokines. Caspase-1 also mediates the inflammatory form of cell death termed pyroptosis, which features the loss of membrane integrity and cell lysis. Caspase-1 cleaves gasdermin D, releasing the N-terminal fragment which forms plasma membrane pores, leading to osmotic lysis.
In vitro, the activation of caspase-1 can be determined by labeling bone marrow-derived macrophages with the caspase-1 activity probe FAM-YVAD-FMK and by labeling the cells with antibodies against the adaptor protein ASC. This technique allows the identification of inflammasome formation and caspase-1 activation in individual cells using fluorescence microscopy. Pyroptotic cell death can be detected by measuring the release of cytosolic lactate dehydrogenase into the medium. This procedure is simple, cost effective and performed in a 96-well plate format, allowing adaptation for screening. In this manuscript, we show that activation of the NLRP3 inflammasome by nigericin leads to the co-localization of the adaptor protein ASC and active caspase-1, leading to pyroptosis.

We describe the detection of NLRP3 inflammasome activation on cellular basis using fluorescence microscopy and staining for active caspase-1 and the adaptor, ASC. A lactate dehydrogenase release assay is presented to detect pyroptotic lysis on a population basis. These techniques can be adapted to study many aspects of inflammasome biology.