건강 연구, 백혈병과 림프종 사회 캐나다와 캐나다의 림프종 재단의 캐나다 연구소에서 교부금에 의해 지원됩니다.

시약 준비 참고 : 모든 PBS는 칼슘 2 +와 MG 2가 포함되어 + <ol><li> 탐지 시약 방 온도를 PBS로 20μM 7 - 아미노 - 4 - chloromethyl coumarin, T - BOC - 류신 - 메티오닌 아미드를 (BOC - LM - CMAC) 추가합니다. 각각의 세포 현탁액은 2mL 탐지 시약을 필요로합니다. </li><li> 1 % Paraformaldehyde (고정 세포) 방 온도를 PBS에 4 % paraformaldehyde 주식 솔루션을 희석. 각 외과 튜브로 나누어지는 1mL 1 % paraformaldehyde. 쓰리 외과 튜브 각 실험 조건이 필요합니다 (예 : 32Dkit, 32Dkit + PD150606, 32Dkit가 + PD98059 9 외과 튜브가 필요) </li><li> PD150606 (calpain 억제제) 100mM의 최종 농도로 DMSO에 PD150606 Reconstitute. 이 시약이 실험 기간 동안 빛으로부터 그것으로 취급 모든 세포를 보호할 수 있습니다. </li><li> PD98059 (MEK1 억제제) 50mM의 최종 농도로 DMSO에 Reconstitute PD98059. </li></ol> 셀 준비 <ol><li> X 10 Opti - 멤 미디어 ML 당 5-1 X 10 6 세포 5 % 태아 소 혈청, IL - 3의 소스 (또는 IL - 3 등에 대한 다른 소스로 WEHI - 3 뜨는와 보충 5 32Dkit 세포를 성장 재조합 단백질 등), 0.1 % 2 - 메르 캅 토 에탄올 항생제. </li><li> 세 15mL 팰컨 튜브 (튜브 당 4 X 10 6 세포)로 12 X 10 6 32Dkit 세포를 수집합니다. </li><li> 15 5 분 1,000 RPM에서 펠렛은 세포 ° C. </li><li> 뜨는을 대기음. 2mL 방 온도 PBS에 세포를 Resuspend. </li><li> 50μM PD150606 하나의 세포 현탁액을 처리합니다. 샘플이 팰컨 튜브 알루미늄 호일로를 덮고하여 빛으로부터 보호됩니다 확인하십시오. 소용돌이는 간단히 다음 어둠 속에서 실온에서 20~30분위한 부화. </li><li> 20μM PD98059 두 번째 세포 현탁액을 처리합니다. 플레이트 37에서 2 시간 동안 35mm 조직 문화 요리와 부화에있는 세포가 ° C. </li></ol> 고정 전지 Calpain 분석 <ol><li> 샘플 PD150606과 잠복기 있지만, 치료 샘플에 calpain 분석을 시작합니다. 각각의 세포 현탁액에 2mL 검출 시약을 추가하여 시작합니다. 즉시 1 % paraformaldehyde (0 분 시점)로 이전 준비 외과 튜브에 세포 현탁액 / 탐지 시약 혼합물의 1mL를 추가합니다. </li><li> 상온에서 5 분 탐지 시약과 셀 suspensions을 품어. 다음 1mL 1 % paraformaldehyde와 외과 튜브에 세포 현탁액의 1mL를 추가합니다. </li><li> 실온에서 추가로 5 분 검출 혼합물로 세포 suspensions을 잠복기 계속합니다. 다음 1mL 1 % paraformaldehyde와 외과 튜브에 세포 현탁액의 1mL를 추가합니다. </li><li> 억제제로 치료했습니다 32Dkit 세포에 calpain 분석을 반복합니다. </li><li> 4 어둠 속에서 야간 세포를 수정 ° C. </li><li> 다음날 펠릿 15 5 분 1,800 RPM에서 세포 ° C. </li><li> 뜨는을 대기음. 750uL PBS에 세포를 Resuspend. 얼음 세포를 놓고 어둠 속에 보관. </li></ol> 고정 셀에 대한 데이터를 수집 <ol><li> LSR II (BD Bioscience)를 사용하여 세포를 분석할 수 있습니다. 필터는 Lightwave Xcyte (UV) 레이저, 여기 라인 355nm 레이저 전력 25mW, 각각 기판 및 제품에 대한 405 및 450 nm의의 방출에 대해 설정됩니다. 기판 및 제품에 대한 밴드 패스 필터 20분의 405과 50분의 450입니다. 기판 및 제품에 대한 긴 패스 필터는 405 450입니다. PMT 전압은 제품의 기판에 대한 350-375과 325-350에 설정되었습니다. </li><li> ; 사이드 분산, 인도 - 1 블루 (로그), 인도 - 1 바이올렛 (로그) 앞으로 흩어 다음 매개 변수 BD FACSDiva 실험 - 설정합니다. 앞으로 흩어 대 라이브 세포 게이트 (그림 1A), 인도 - 1 블루 히스토그램, 인도 - 1 바이올렛 히스토그램, 인도 - 1 블루 대 인도 - 1 바이올렛과 ​​사이드 분산형 점 줄거리 : 워크시트에 다음과 같은 그래프를 설정 도트 플롯. </li><li> LSR AlignFlow과 AlignFlow와 II 플러스 형광 구슬 (Invitrogen)를 보정합니다. PMT 전압 전에 모든 실험에 동일한 채널에서 비드 형광 히스토그램의 절정을 배치 조정해야합니다. </li><li> 0 분 시점에서 32Dkit 세포를 사용하여 데이터를 수집 시작합니다. 필요에 따라 매개 변수 전압을 조정합니다. 수집 및 샘플 10,000 이벤트를 기록합니다. </li><li> 각 튜브 사이 외과 버퍼와 함께 시스템을 rinsing, 각 샘플에 대한 인수를 반복합니다. </li><li> 데이터를 내보낼 수 있습니다. 확인 LSR II는 기관의 지침에 따라 청소합니다. </li></ol> 라이브 세포 Calpain 분석 <ol><li> 노 PD150606와 위의 세포 suspensions를 준비합니다. LSR II에 샘플을 가지고 이상과 같은 필터와 파라미터를 설정합니다. 위와 같이 워크시트를 설정합니다. 인도 - 1 블루 대 시간을 보여주는 대형 도트 플롯을 포함합니다. 최대 NUM를 얻기위한 프로그램을 설정이벤트 BER (NO 중지 게이트). </li><li> 32Dkit 세포의 50μM PD150606 하나의 세포 현탁액을 추가합니다. 어둠의 샘플 보관 20-30 명의 분 실온에서 품어. </li><li> 노 PD150606과 32Dkit 세포 현탁액에 대한 데이터를 수집 시작합니다. 초당 200-300 이벤트의 낮은 유속을 사용합니다. 30 초 일분 후 시스템에서 튜브를 제거합니다. 세포에 20μM BOC - LM - CMAC을 추가합니다. 소용돌이는 간단히 다음 데이터를 획득 계속합니다. </li><li> 10~20분 또는 calpain 활동 고원까지 데이터를 수집. </li><li> PD150606 취급 32Dkit 세포와 3 단계와 4 단계를 반복합니다. </li><li> 데이터를 내보낼 수 있습니다. 확인 LSR II는 기관의 지침에 따라 청소합니다. </li></ol> 콤플렉스 (혼합) 셀 인구의 Calpain 분석 *이 실험은 마우스 비장에서 수확 세포 현탁액에 32Dkit 세포를 식별합니다. <ol><li> 각 마우스의 비장을 수확. 실온에서 10 분 NH 4 Club 호텔과 적혈구 세포를 Lyse. </li><li> 2mL PBS의 단세포 suspensions를 준비합니다. </li><li> 두 각각의 세포 현탁액을 나눕니다. 어둠 속에서 실온에서 20-30분에 대한 50μM PD150606 1 세포 현탁액을 처리합니다. </li><li> 고정 셀에 Calpain 어세이 (1-6 단계)에 대한 위에서 진행 </li><li> 뜨는을 대기음. 500μL PBS에 세포를 씻으십시오. 뜨는을 대기음. </li><li> 2 % BSA와 200μL 얼룩 버퍼에있는 세포를 Resuspend. 실온에서 15 분 동안 품어. </li><li> 기본 항체 (FITC 안티 마우스 CD117)를 준비합니다. 항체는 버퍼를 얼룩의 1:200 희석에 사용해야합니다. 항체가 어둠에 갇혀있다 확인하십시오. </li><li> 세포에 200μL 기본 항체를 추가합니다. 어둠 속에서 실온에서 30 분 알을 품다. </li><li> 유동세포계측법의 볼륨을 증가 400μL PBS를 추가합니다. 얼음에 세포를 놓고 어둠 속에 보관. </li><li> 고정 셀에 대한 데이터를 취득에서 설명한대로 진행합니다. 매개 변수 FITC 추가하고 워크시트에서 FITC에 대한 히스토그램을 포함합니다. </li></ol> 분석 및 대표 결과 <ol><li> FlowJo (Treestar)를 사용하여 데이터를 분석할 수 있습니다. 순방향 및 측면 산란 프로파일 (그림 1A)에 따라 사는 세포 게이트. 가능한 세포 게이트 (그림 1B)에서 인도 블루에 대한 의미 형광을 확인합니다. </li><li> 혼합 셀 인구) <ul><li> 32Dkit 인구를 식별 histograms 및 도트 플롯에 FITC 형광을 사용하여 </li><li> 32Dkit 인구의 인도 블루에 대한 의미 형광을 결정 </li></ul></li><li> 그래프 평균 형광 (그림 2A)을 위해 Microsoft Excel을 사용하십시오. 5 10 분 사이의 라인의 기울기를 계산. 그래프 기울기 (그림 2B) 셀에 calpain 활동을 결정하기 위해 막대 그래프로. </li></ol><img src="/files/ftp_upload/2050/2050fig1a.jpg" alt="그림 1A" /> 그림 1A. 순방향 및 측면 산란 프로파일을 기반으로 살 세포를 결정. <img src="/files/ftp_upload/2050/2050fig1b.jpg" alt="그림 1B" /> 그림 1B는. 10 분 시간 코스 이상의 형광 변화를 의미합니다. <img src="/files/ftp_upload/2050/2050fig2a.jpg" alt="그림 2A" /> 의미 형광의 그림 2A가. 대표 그래프. <img src="/files/ftp_upload/2050/2050fig2b.jpg" alt="그림 2B" /> 억제제와 32Dkit 세포 그림 2B. Calpain 활동.

<ol>
	<li>Goll, D. E., Thompson, V. F., Li, H., Wei, W., Cong, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Calpain System. Physiol. Rev. 83, 731-801 (2003).</li><li>Niapour, M., Berger, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+Cytometric+Measurement+of+Calpain+Activity+in+Living+Cells.">Flow Cytometric Measurement of Calpain Activity in Living Cells.</a> Cytometry A. 71A, 475-485 (2007).</li><li>Leloup, L., Daury, L., Maz&#233;res, G., Cottin, P. <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+ERK/MAP+kinase+signalling+pathway+in+milli-calpain+activation+and+myogenic+cell+migration.">Involvement of the ERK/MAP kinase signalling pathway in milli-calpain activation and myogenic cell migration.</a> The International Journal of Biochemistry and Cell Biology. 39, 1177-1189 (2007).</li></ol>

관심 없음 충돌 선언하지 않습니다.

고정 생활 32Dkit 세포의 calpain 활동이 비디오 프로토콜에서 결정되었습니다. calpain 억제제 PD150606과 세포 라인의 치료는 세포의 calpain 활동의 완전한 억제 결과. 또한, MEK1 억제제 PD98059 치료는 세포의 calpain 활동의 완전한 억제 결과. ERK는 calpain - 2 3의 주요 활성입니다. 이러한 결과는 calpain - 2 활동이 32Dkit 세포의 지배는 것이 좋습니다. 우리 연구실에서 현재 작업 증가 calpain 활동의 결과와...

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		<th>Material Name</th>
		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>PBS BOC-LM-CMAC 4% paraformaldehyde</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>&nbsp;</td>
<td> Dissolve 4g paraformaldehyde in 100mL PBS. In fume hood, heat sample with stirring until clear. Let cool then store at 4&deg;C</td>
</tr>
<tr>
<td>PD150606</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>&nbsp;</td>
<td>Use freshly reconstituted PD150606 for maximal calpain inhibition.</td>
</tr>
<tr>
<td>PD98059</td>
<td>&nbsp;</td>
<td>Cell Signaling Technology</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>AlignFlow, AlignFlow plus fluorescent beads FACS tubes</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>LSR II</td>
<td>&nbsp;</td>
<td>BD Bioscience</td>
<td>&nbsp;</td>
<td> Supernatant of WEHI-3 cells is used as a source of IL-3 in these experiments. Each collection of WEHI-3 supernatant is titrated to determine the optimal concentration used for 32Dkit cell growth. </td>
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measuring calpain activity in fixed and living cells by flow cytometry

Calpains는 유비 쿼터스 세포, 칼슘 구분, 중립 시스테인 프로 테아제 아르<sup&gt; 1</sup&gt;. Calpains는 신호, cytoskeletal 리모델링, 유전자 발현, apoptosis의 조절 및 세포주기의 진행 등 많은 생리 과정에서 중요한 역할을<sup&gt; 1</sup&gt;. Calpains는 근육질 dystrophies, 암, 당뇨병, 알츠하이머 질환과 다중 경화증 등 많은 pathologies에 연루되었습니다<sup&gt; 1</sup&gt;. Calpain 규정은 복잡하고 불완전하게 이해된다. mRNA와 단백질 수준 calpain 활동을 측정하는 유전자 또는 단백질 표현 기법의 사용을 제한, 활동이 저조한 상관 관계. 이 비디오 프로토콜 세부 사항은 고정 및 살아있는 세포에서 calpain 활동을 측정하기위한 우리 연구실에 흐름 cytometric 분석을 개발했습니다. 이 방법은 calpain에 의해 구체적으로 죽습니다 형광 기판 BOC - LM - CMAC은, calpain 활동을 측정합니다.<sup&gt; 2</sup&gt;이 비디오에서는 고정 생활 murine 32Dkit의 백혈병 세포, 혼자 또는 splenocyte 인구의 일부로 calpain 활동 LSRII (BD Bioscience)를 사용하여 측정됩니다. 32Dkit 전지는 고가 활동이 정상 splenocytes에 비해해야 표시됩니다.

Watch this Scientific Journal Video about Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry at JoVE.com

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

이 문서 유동세포계측법를 사용하여 고정하고 살아있는 세포에서 calpain 활동을 측정하기위한 세부 프로토콜을 것입니다.

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, ...

measuring-calpain-activity-in-fixed-and-living-cells-by-flow-cytometry

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JoVE Journal

Immunology and Infection

11.8K 조회수.  University of Toronto. 이 문서 유동세포계측법를 사용하여 고정하고 살아있는 세포에서 calpain 활동을 측정하기위한 세부 프로토콜을 것입니다.

비디오: Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response following an infection. Despite efforts to identify unique surface marker on Tregs, the only unique feature is their ability to suppress the proliferation and function of effector T cells. While it is clear that only in vitro assays can be used in assessing human Treg function, this becomes problematic when assessing the results from cross-sectional studies where healthy cells and cells isolated from subjects with autoimmune diseases (like Type 1 Diabetes-T1D) need to be compared. There is a great variability among laboratories in the number and type of responder T cells, nature and strength of stimulation, Treg:responder ratios and the number and type of antigen-presenting cells (APC) used in human in vitro suppression assays. This variability makes comparison between studies measuring Treg function difficult. The Treg field needs a standardized suppression assay that will work well with both healthy subjects and those with autoimmune diseases. We have developed an in vitro suppression assay that shows very little intra-assay variability in the stimulation of T cells isolated from healthy volunteers compared to subjects with underlying autoimmune destruction of pancreatic &beta;-cells. The main goal of this piece is to describe an in vitro human suppression assay that allows comparison between different subject groups. Additionally, this assay has the potential to delineate a small loss in nTreg function and anticipate further loss in the future, thus identifying subjects who could benefit from preventive immunomodulatory therapy1. Below, we provide thorough description of the steps involved in this procedure. We hope to contribute to the standardization of the in vitro suppression assay used to measure Treg function. In addition, we offer this assay as a tool to recognize an early state of immune imbalance and a potential functional biomarker for T1D.

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Tregs are potent suppressors of the immune system. There is a lack of unique surface markers to define them, hence, definitions of Tregs are primarily functional. Here we describe an optimized in vitro assay capable of identifying immune imbalance in subjects at risk to develop T1D.

사람의 체외에서</em면역 불균형의 초기 국가의 인정을위한 심사 도구를> 억제

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response ...

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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 ...

Examination of Thymic Positive and Negative Selection by Flow Cytometry

A healthy immune system requires that T cells respond to foreign antigens while remaining tolerant to self-antigens. Random rearrangement of the T cell receptor (TCR) &alpha; and &beta; loci generates a T cell repertoire with vast diversity in antigen specificity, both to self and foreign. Selection of the repertoire during development in the thymus is critical for generating safe and useful T cells. Defects in thymic selection contribute to the development of autoimmune and immunodeficiency disorders1-4. 
T cell progenitors enter the thymus as double negative (DN) thymocytes that do not express CD4 or CD8 co-receptors. Expression of the &alpha;&beta;TCR and both co-receptors occurs at the double positive (DP) stage. Interaction of the &alpha;&beta;TCR with self-peptide-MHC (pMHC) presented by thymic cells determines the fate of the DP thymocyte. High affinity interactions lead to negative selection and elimination of self-reactive thymocytes. Low affinity interactions result in positive selection and development of CD4 or CD8 single positive (SP) T cells capable of recognizing foreign antigens presented by self-MHC5.
Positive selection can be studied in mice with a polyclonal (wildtype) TCR repertoire by observing the generation of mature T cells. However, they are not ideal for the study of negative selection, which involves deletion of small antigen-specific populations. Many model systems have been used to study negative selection but vary in their ability to recapitulate physiological events6. For example, in vitro stimulation of thymocytes lacks the thymic environment that is intimately involved in selection, while administration of exogenous antigen can lead to non-specific deletion of thymocytes7-9. Currently, the best tools for studying in vivo negative selection are mice that express a transgenic TCR specific for endogenous self-antigen. However, many classical TCR transgenic models are characterized by premature expression of the transgenic TCR&alpha; chain at the DN stage, resulting in premature negative selection. Our lab has developed the HYcd4 model, in which the transgenic HY TCR&alpha; is conditionally expressed at the DP stage, allowing negative selection to occur during the DP to SP transition as occurs in wildtype mice10.
Here, we describe a flow cytometry-based protocol to examine thymic positive and negative selection in the HYcd4 mouse model. While negative selection in HYcd4 mice is highly physiological, these methods can also be applied to other TCR transgenic models. We will also present general strategies for analyzing positive selection in a polyclonal repertoire applicable to any genetically manipulated mice.

We present a flow cytometry-based method to examine T cell development in vivo using genetically manipulated mice on a wildtype or T cell receptor transgenic background.

유동 세포 계측법에 의한 Thymic 긍정적이고 부정적인 선택의 시험

A healthy immune system requires that T cells respond to foreign  antigens while remaining tolerant to self-antigens. Random rearrangement of the  T cell ...

Cell-based Flow Cytometry Assay to Measure Cytotoxic Activity

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells to kill CD4+ T cells loaded with their cognate peptide. Target CD4+ T cells are divided into two populations, labeled with two different concentrations of CFSE. One population is pulsed with the peptide of interest (CFSE-low) while the other remains un-pulsed (CFSE-high). Pulsed and un-pulsed CD4+ T cells are mixed at an equal ratio and incubated with an increasing number of purified CD8+ T cells. The specific killing of autologous target CD4+ T cells is analyzed by flow cytometry after coculture with CD8+ T cells containing the antigen-specific effector CD8+ T cells detected by peptide/MHCI tetramer staining. The specific lysis of target CD4+ T cells measured at different effector versus target ratios, allows for the calculation of lytic units, LU30/106 cells. This simple and straightforward assay allows for the accurate measurement of the intrinsic capacity of CD8+ T cells to kill target CD4+ T cells.

This protocol describes a sensitive, cell-based cytotoxicity assay. By enumerating the decrease in frequency of live target CD4+ T cells in the presence of an increasing number of effector CD8+ T cells, this assay allows for the direct assessment of cytolytic activity of antigen-specific CD8+ T cells.

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells ...

Measuring Attachment and Internalization of Influenza A Virus in A549 Cells by Flow Cytometry

Attachment to target cells followed by internalization are the very first steps of the life cycle of influenza A virus (IAV). We provide here a detailed protocol for measuring relative changes in the amount of viral particles that attach to A549 cells, a human lung epithelial cell line, as well as in the amount of particles that are internalized into the cell. We use biotinylated virus which can be easily detected following staining with Cy3-labeled streptavidin (STV-Cy3). We describe the growth, purification and biotinylation of A/WSN/33, a widely used IAV laboratory strain. Cold-bound biotinylated IAV particles on A549 cells are stained with STV-Cy3 and measured using flow cytometry. To investigate uptake of viral particles, cold-bound virus is allowed to internalize at 37 &#176;C. In order to differentiate between external and internalized viral particles, a blocking step is applied: Free binding spots on the biotin of attached virus on the cell surface are bound by unlabeled streptavidin (STV). Subsequent cell permeabilization and staining with STV-Cy3 then enables detection of internalized viral particles. We present a calculation to determine the relative amount of internalized virus. This assay is suitable to measure effects of drug-treatments or other manipulations on attachment or internalization of IAV.

We present a protocol describing a semi-quantitative method for measuring both, the attachment of influenza A virus to A549 cells, as well as the internalization of virus particles into the target cells by flow cytometry.

유동 세포 계측법에 의해 A549 세포의 부착 및 국제화 인플루엔자의 바이러스를 측정

Attachment to target cells followed by internalization are the very first steps of the life cycle of influenza A virus (IAV). We provide here a detailed ...

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

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method enables researchers to better understand the characteristics of a single cell population without the influence of other cells. Compared to other methods of cell enrichment, such as magnetic-activated cell sorting (MCS), FACS is more flexible and accurate for cell separation due to the ability of phenotype detection by flow cytometry. In addition, FACS is usually capable of separating multiple cell populations simultaneously, which improves the efficiency and diversity of experiments. Although FACS has some limitations, it has been broadly used to purify cells for functional studies in both in vitro and in vivo settings. Here we report a protocol using fluorescence-activated cell sorting to isolate a very rare population of immune cells, plasmacytoid dendritic cells (pDC), with high purity from the bone marrow of lupus-prone mice for in vitro functional studies of pDC.

We report a protocol using fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells (pDC) with high purity from the bone marrow of lupus-prone mice for functional studies of pDC.

마우스 골수에서 형질 수지상 세포의 정화를위한 형광 활성화 셀 정렬

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

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

고립과 Murine 인간과 치경 대 식 세포의 생체 외에서 문화

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Preparation of Whole Bone Marrow for Mass Cytometry Analysis of Neutrophil-lineage Cells

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a myeloid-biased 39-antibody CyTOF panel to evaluate the hematopoietic system with a focus on the neutrophil-lineage cells by using this protocol. The CyTOF result was analyzed with an open-resource dimensional reduction algorithm, viSNE, and the data was presented to demonstrate the outcome of this protocol. We have discovered new neutrophil-lineage cell populations based on this protocol. This protocol of fresh whole BM preparation may be used for 1), CyTOF analysis to discover unidentified cell populations from whole BM, 2), investigating whole BM defects for patients with blood disorders such as leukemia, 3), assisting optimization of fluorescence-activated flow cytometry protocols that utilize fresh whole BM.

Here, we present a protocol to process fresh bone marrow (BM) isolated from mouse or human for high-dimensional mass cytometry (Cytometry by Time-Of-Flight, CyTOF) analysis of neutrophil-lineage cells.

호중구 계보 세포의 질량 세포 분석 분석을위한 전체 골수의 준비

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a ...

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are cleared from the human lung alveoli by being phagocytosed by innate monocytes and/or neutrophils. This protocol offers a fast and reliable measurement of phagocytosis by flow cytometry using fluorescein isothiocyanate (FITC)-labeled conidia for co-incubation with human leukocytes and subsequent counterstaining with an anti-FITC antibody to allow discrimination of internalized and cell-adherent conidia. Major advantages of this protocol are its rapidness, the possibility to combine the assay with cytometric analysis of other cell markers of interest, the simultaneous analysis of monocytes and neutrophils from a single sample and its applicability to other cell wall-bearing fungi or bacteria. Determination of percentages of phagocytosing leukocytes provides a means to microbiologists for evaluating virulence of a pathogen or for comparing pathogen wildtypes and mutants as well as to immunologists for investigating human leukocyte capabilities to combat pathogens.

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

This protocol provides a fast and reliable method to quantitatively measure phagocytosis of Aspergillus fumigatus conidia by human primary phagocytes using flow cytometry and to discriminate phagocytosis of conidia from mere adhesion to leukocytes.

유세포 측정을 사용하여 인간 백혈구에 의한 아스퍼질러스 훈증 코니디아의 식세포증 측정

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are ...

Measuring Erythrocyte Complement Receptor 1 Using Flow Cytometry

CR1 (CD35, Complement Receptor type 1 for C3b/C4b) is a high molecular weight membrane glycoprotein of about 200 kDa that controls complement activation, transports immune complexes, and participates in humoral and cellular immune responses. CR1 is present on the surface of many cell types, including erythrocytes, and exhibits polymorphisms in length, structure (Knops, or KN, blood group), and density. The average density of CR1 per erythrocyte (CR1/E) is 500 molecules per erythrocyte. This density varies from one individual to another (100&#8211;1,200 CR1/E) and from one erythrocyte to another in the same individual. We present here a robust flow cytometry method to measure the density of CR1/E, including in subjects expressing a low density, with the help of an amplifying immunostaining system. This method has enabled us to show the lowering of CR1 erythrocyte expression in diseases such as Alzheimer&#39;s disease (AD), systemic lupus erythematosus (SLE), AIDS, or malaria.

The aim of this method is to determine the CR1 density in the erythrocytes of any subject by comparing with three subjects whose erythrocyte CR1 density is known. The method uses flow cytometry after immunostaining of the subjects&#39; erythrocytes by an anti-CR1 monoclonal antibody coupled to an amplified system using phycoerythrin (PE).

유세포 분석을 사용하여 적혈구 보체 1 보체 측정

CR1 (CD35, Complement Receptor type 1 for C3b/C4b) is a high molecular weight membrane glycoprotein of about 200 kDa that controls complement activation, ...