이 연구는 웨스턴 켄터키 대학에서 학부 장학금위원회에서 새로운 교수 부여에 의해 지원되었다.

1. 손가락 - 스틱 혈액 샘플링 <ol><li> 전체 혈액의 컬렉션은 일반적으로의 정지 다음 (일반적으로 1~3시간에서 동안 기준 측정 (보통 10-15 분 앉아 휴식을 다음) 또는 즉시 운동 시합에 따라, 그리고 사후 운동 기간 동안 휴식을 취해 운동). </li><li> 유니버설주의를 사용하면, 먼저 70% 이소 프로필 알코올로 손가락을 소독. </li><li> 다음 찔린 자동 첨두 또는 유사한 장치를 사용하여 사이트입니다. 우리가 주제 편안함을 제공하는 30 게이지 랜싯를 사용하지만, 더 큰 게이지 주사 바늘을 이용 할 수 있습니다. </li><li> 혈액의 첫 번째 드롭을 닦아 후 응고 방지하기 위해 리튬 헤파린 취급 모세관 튜브 또는 microvettes에 혈액을 수집합니다. </li></ol> 2. 세포 Phenotyping 및 Apoptosis의 스테 이닝 <ol><li> 250 μL 바인딩 버퍼에 titred 항체 패널 (표 1 배선도 참조) 10 μL heparinized 전체 혈액을 추가합니다. 우리는 성공을 1시 20분 희석 계수를 사용해야하지만, 각각의 실험실이 최고의 작업 솔루션을 결정하기 위해 시약을 titre해야합니다. 백혈구 집합에서 운동 유발성 apoptosis의 결정에 대해 표 1. 항체 패널. 늦은 apoptosis가 Annexin V 7 - AAD 더블 긍정적인 상표에 의해 정의된 동안 조기 apoptosis는 Annexin V의 표현에 의해 정의되었다. 괴사 세포가 V - 7 - AAD + 세포에만 Annexin했다. <table border="1"><tbody><tr><td> 튜브 1 </td><td> 튜브 2 </td><td> 관 세 </td><td> 튜브 사 </td></tr><tr><td> 세포는 관 </td><td> Annexin V - FITC </td><td> Annexin V - FITC </td><td> Annexin V - FITC </td></tr><tr><td></td><td> 안티 인간 CD4 - PE </td><td> 안티 인간 CD8 - PE </td><td> 안티 인간 CD19 - PE </td></tr><tr><td></td><td> 7 - AAD </td><td> 7 - AAD </td><td> 7 - AAD </td></tr><tr><td></td><td> 안티 인간 CD45RA - APC </td><td> 안티 인간 CD45RA - APC </td><td></td></tr></tbody></table> 7 - AAD = 7 - 아미노 actinomycin D, APC = Allophycocyanin, CD4 = 도우미 T의 lymphocytes, CD8 = 세포 독성 / 억제 T의 lymphocytes, CD19 = B lymphocytes, CD45RA = 순진한 세포 하위 집합, FITC = 플루오레신 isothiocyanate, PE = Phycoerythrin. </li><li> 30 분 어두운 실온에서 알을 품다. </li><li> 50~10분 위해 1075 XG에 원심 분리기. </li><li> 가만히 따르다가 300 μL 적혈구 세포 용해 완충액을 추가하고, 철저히 소용돌이. </li><li> 15 분 동안 상온에서 알을 품다. </li><li> RBC의 용해 반응을 중지 300 μL PBS를 추가합니다. </li><li> 50~10분 위해 1075 XG에 원심 분리기. </li><li> 가만히 따르다, 랙, 50 μL 구속력 버퍼를 추가합니다. </li><li> 유동세포계측법으로 분석합니다. </li></ol> 전용 튜브 배경 autofluorescence를 감지에 부정적인 제어 역할을하는 세포를, 그리고 각 개별가 포함된 튜브의 흐름 cytometer의 설정 초기 때 보상을 설정 fluorochrome : 이것은 최소한 다음과 같은 컨트롤을 활용하는 것이 좋습니다. 또한, 우리는 실험을 설정 성공적으로 보상 기준 비즈를 사용합니다.

<ol>
	<li>Simpson, R. J., Florida-James, G. D., Whyte, G. P., Black, J. R., Ross, J. A., Guy, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptosis+does+not+contribute+to+the+blood+lymphocytopenia+observed+after+intensive+and+downhill+treadmill+running+in+humans.">Apoptosis does not contribute to the blood lymphocytopenia observed after intensive and downhill treadmill running in humans.</a> Res. Sports Med. 15, 157-174 (2007).</li><li>Steensberg, A., Morrow, J., Toft, A. D., Bruunsgaard, H., Pedersen, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prolonged+exercise,+lymphocyte+apoptosis+and+F2-isoprostanes.">Prolonged exercise, lymphocyte apoptosis and F2-isoprostanes.</a> Eur. J. Appl. Physiol. 87, 38-42 (2002).</li><li>Mooren, F. C., Bloming, D., Lechtermann, A., Lerch, M. M., V&#246;lker, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphocyte+apoptosis+after+exhaustive+and+moderate+exercise.">Lymphocyte apoptosis after exhaustive and moderate exercise.</a> J. Appl. Physiol. 93, 147-153 (2002).</li><li>Peters, E. M., Eden, M. V. a. n., Tyler, N., Ramautar, A., Chutergoon, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prolonged+exercise+does+not+cause+lymphocyte+DNA+damage+or+increased+apoptosis+in+well-trained+endurance+athletes.">Prolonged exercise does not cause lymphocyte DNA damage or increased apoptosis in well-trained endurance athletes.</a> Eur. J. Appl. Physiol. 98, 124-131 (2006).</li><li>Wang, J. -. S., Huang, Y. -. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+exercise+intensity+on+lymphocyte+apoptosis+induced+by+oxidative+stress+in+men.">Effects of exercise intensity on lymphocyte apoptosis induced by oxidative stress in men.</a> Eur. J. Appl. Physiol. 95, 290-291 (2005).</li><li>Mooren, F. C., Lechtermann, A., V&#246;lker, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exercise-induced+apoptosis+of+lymphocytes+depends+on+training+status.">Exercise-induced apoptosis of lymphocytes depends on training status.</a> Med. Sci. Sports Exerc. 36, 1476-1483 (2004).</li><li>Navalta, J. W., Sedlock, D. A., Park, K. -. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+treatment+influences+the+yield+of+apoptotic+lymphocytes+after+maximal+exercise.">Blood treatment influences the yield of apoptotic lymphocytes after maximal exercise.</a> Med. Sci. Sports Exerc. 37, 369-373 (2005).</li></ol>

관심 없음 충돌 선언하지 않습니다.

법으로서 샘플 수집 과정 및 후속 분석 운동 유발성 apoptosis 림프구의 조기 늦게 두 단계의 분석에 대한 증명입니다. 그것은 명확하게 괴사 및 apoptotic되지 않은 그 세포를 제외하는 것도 가능합니다. 이 절차 동안, 이전에 여러 시간 지점에서 antecubital 지역에서 침략 venipuncture와 관련된 단점과 불편을 극복하고, 즉시 운동 후 또는 운동 시합 기간 동안 카테터를 확보. 조사의 측면에서, 이러한 절차?...

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<td>Flow Cytometry Binding Buffer</td>
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<td>eBioscience</td>
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<td>RBC Lysis Buffer</td>
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<td>eBioscience</td>
<td>00-4333-57</td>
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<td>Lancet Device</td>
<td>Accu Check Soft Touch</td>
<td>Roche Diagnostics Corp.</td>
<td>&nbsp;</td>
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<td>Capillary tubes</td>
<td>Heparinized Capillary Tubes</td>
<td>Chase Scientific Glass, Inc.</td>
<td>501</td>
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<td>Centrifuge</td>
<td>GmC Lab</td>
<td>Gilson</td>
<td>PMS-880</td>
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<td>Annexin V-FITC Conjugated</td>
<td>&nbsp;</td>
<td>Biovision Inc.</td>
<td>K201-400-1</td>
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<td>CD4-PE Conjugated</td>
<td>Anti-human, clone RPA-T4</td>
<td>eBioscience</td>
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<td>CD8-PE Conjugated</td>
<td>Anti-human, clone OKT8</td>
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<td>Anti-human, clone HI100</td>
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<td>Flow cytometer</td>
<td>C6</td>
<td>Accuri</td>
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<td>&nbsp;</td>		</tbody>
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finger-stick blood sampling methodology for the determination of exercise-induced lymphocyte apoptosis

운동은 면역 세포의 apoptosis를 유도하는 수있는 생리적 자극이다. 지금까지 다양한 제한은 특히 이전에 세포 죽음의 평가에 혈액 샘플을 분리하고 치료하는 데 필요한 시간의 양을에 관한,이 현상의 측정과 확인되었습니다. 이 때문에 면역 세포 apoptosis에보고 증가가 시스템에 대한 운동의 실질적인 효과에 기여하거나, 결국이 측정을 얻기 위해 필요한 시간과 처리를 반영 아르 될 수 있는지 여부를 확인하기가 어렵습니다. 이 문서에서 우리는 운동 유발성 apoptosis 림프구의 분석을위한 신속하고 법으로서 절차를 보여줍니다. 다른 기법과는 달리, 전체 혈액 샘플을 취득 즉시 항체 패널에 추가됩니다. 잠복기에 따라, 적혈구는 lysed 및 샘플 분석 준비입니다. 손가락 - 스틱 샘플링 절차의 사용은 필요한 혈액의 볼륨을 감소하고, 주제에 대한 불편을 최소화합니다.

Watch this Scientific Journal Video about Finger-stick Blood Sampling Methodology for the Determination of Exercise-induced Lymphocyte Apoptosis at JoVE.com

Finger-stick Blood Sampling Methodology for the Determination of Exercise-induced Lymphocyte Apoptosis

운동은 면역 세포의 apoptosis를 유도 할 수있다. 다양한 측정 한계는 특히 사전 심사에 혈액 샘플을 분리하고 치료하는 데 필요한 시간의 양을 관련된있다. 보여주는 운동 유발성 림프구의 apoptosis의 분석을위한 신속하고 법으로서 절차입니다.

운동 유발성 Apoptosis 림프구의 결정을위한 손가락 - 스틱 혈액 샘플링 방법론

Exercise is a physiological stimulus capable of inducing apoptosis in immune cells. To date, various limitations have been identified with the measurement ...

finger-stick-blood-sampling-methodology-for-determination-exercise

Research

JoVE Journal

Immunology and Infection

12.1K 조회수.  Western Kentucky University. 운동은 면역 세포의 apoptosis를 유도 할 수있다. 다양한 측정 한계는 특히 사전 심사에 혈액 샘플을 분리하고 치료하는 데 필요한 시간의 양을 관련된있다. 보여주는 운동 유발성 림프구의 apoptosis의 분석을위한 신속하고 법으로서 절차입니다.

비디오: Finger-stick Blood Sampling Methodology for the Determination of Exercise-induced Lymphocyte Apoptosis

Depletion of Specific Cell Populations by Complement Depletion

The purification of immune cell populations is often required in order to study their unique functions. In particular, molecular approaches such as real-time PCR and microarray analysis require the isolation of cell populations with high purity. Commonly used purification strategies include fluorescent activated cell sorting (FACS), magnetic bead separation and complement depletion. Of the three strategies, complement depletion offers the advantages of being fast, inexpensive, gentle on the cells and a high cell yield. The complement system is composed of a large number of plasma proteins that when activated initiate a proteolytic cascade culminating in the formation of a membrane-attack complex that forms a pore on a cell surface resulting in cell death1. The classical pathway is activated by IgM and IgG antibodies and was first described as a mechanism for killing bacteria. With the generation of monoclonal antibodies (mAb), the complement cascade can be used to lyse any cell population in an antigen-specific manner. Depletion of cells by the complement cascade is achieved by the addition of complement fixing antigen-specific antibodies and rabbit complement to the starting cell population. The cells are incubated for one hour at 37&deg;C and the lysed cells are subsequently removed by two rounds of washing. MAb with a high efficiency for complement fixation typically deplete 95-100% of the targeted cell population. Depending on the purification strategy for the targeted cell population, complement depletion can be used for cell purification or for the enrichment of cell populations that then can be further purified by a subsequent method.

To effectively study the function of immune cell populations their purification is often required. Complement depletion is a fast and inexpensive technique for the isolation of immune cell populations with high purity.

보완 고갈에 의해 특정 세포 집단의 고갈

The purification of immune cell populations is often required in order to study their unique functions.  In particular, molecular approaches such as ...

연구

면역학 및 감염병학

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS)

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known phenotype.  Other bulk methods of purification include panning, complement depletion and magnetic bead separation.  However, FACS has several advantages over other available methods.  FACS is the preferred method when very high purity of the desired population is required, when the target cell population expresses a very low level of the identifying marker or when cell populations require separation based on differential marker density.  In addition, FACS is the only available purification technique to isolate cells based on internal staining or intracellular protein expression, such as a genetically modified fluorescent protein marker.  FACS allows the purification of individual cells based on size, granularity and fluorescence.  In order to purify cells of interest, they are first stained with fluorescently-tagged monoclonal antibodies (mAb), which recognize specific surface markers on the desired cell population (1).  Negative selection of unstained cells is also possible.  FACS purification requires a flow cytometer with sorting capacity and the appropriate software.  For FACS, cells in suspension are passed as a stream in droplets with each containing a single cell in front of a laser.  The fluorescence detection system detects cells of interest based on predetermined fluorescent parameters of the cells.  The instrument applies a charge to the droplet containing a cell of interest and an electrostatic deflection system facilitates collection of the charged droplets into appropriate collection tubes (2).  The success of staining and thereby sorting depends largely on the selection of the identifying markers and the choice of mAb.  Sorting parameters can be adjusted depending on the requirement of purity and yield.  Although FACS requires specialized equipment and personnel training, it is the method of choice for isolation of highly purified cell populations.

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting FACS

For many scientific studies requiring a biological and chemical analysis of cell populations the cells must be in a high state of purity. Fluorescence activated cell sorting (FACS) is a superior method in which to obtain pure cell populations.

형광 활성 세포 정렬 (외과)에 의해 특정 세포 인구의 정제

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known ...

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular matrix proteins. The precise spatiotemporal activation of integrins from a low affinity state to a high affinity state at the cell leading edge is important for T lymphocyte migration 1. Likewise, retraction of the cell trailing edge, or uropod, is a necessary step in maintaining persistent integrin-dependent T lymphocyte motility 2. Many therapeutic approaches to autoimmune or inflammatory diseases target integrins as a means to inhibit the excessive recruitment and migration of leukocytes 3. To study the molecular events that regulate human T lymphocyte migration, we have utilized an in vitro system to analyze cell migration on a two-dimensional substrate that mimics the environment that a T lymphocyte encounters during recruitment from the vasculature. T lymphocytes are first isolated from human donors and are then stimulated and cultured for seven to ten days. During the assay, T lymphocytes are allowed to adhere and migrate on a substrate coated with intercellular adhesion molecule-1 (ICAM-1), a ligand for integrin LFA-1, and stromal cell-derived factor-1 (SDF-1). Our data show that T lymphocytes exhibit a migratory velocity of ~15 &mu;m/min. T lymphocyte migration can be inhibited by integrin blockade 1 or by inhibitors of the cellular actomyosin machinery that regulates cell migration 2.

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

T lymphocyte migration occurs during homing to lymphoid organs, exit from the vasculature, and entering into peripheral tissues. Here, we describe a protocol that can be used to analyze T lymphocyte migration in vitro.

마이 그 레이션의 인간 T 림프구 분리, 문화 및 분석 체외에서</em

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular ...

The Use of Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE) to Monitor Lymphocyte Proliferation

Carboxyfluorescein succinimidyl ester (CFSE) is an effective and popular means to monitor lymphocyte division1-3. CFSE covalently labels long-lived intracellular molecules with the fluorescent dye, carboxyfluorescein. Thus, when a CFSE-labeled cell divides, its progeny are endowed with half the number of carboxyfluorescein-tagged molecules and thus each cell division can be assessed by measuring the corresponding decrease in cell fluorescence via Flow cytometry. The capacity of CFSE to label lymphocyte populations with a high fluorescent intensity of exceptionally low variance, coupled with its low cell toxicity, make it an ideal dye to measure cell division. Since it is a fluorescein-based dye it is also compatible with a broad range of other fluorochromes making it applicable to multi-color flow cytometry. This article describes the procedures typically used for labeling mouse lymphocytes for the purpose of monitoring up to 8 cell divisions. These labeled cells can be used both for in vitro and in vivo studies.

The Use of Carboxyfluorescein Diacetate Succinimidyl Ester CFSE to Monitor Lymphocyte Proliferation

CFSE covalently labels long-lived intracellular molecules with the fluorescent dye, carboxyfluorescein. As such, when a CFSE-labeled cell divides, its progeny have half the amount of fluorescence, which can thereby be used to assess cell division. This article describes the procedures typically used for labeling mouse lymphocytes with CFSE.

림프구 확산을 모니터 Carboxyfluorescein 디아 세 테이트 Succinimidyl 에스터 (CFSE)의 사용

Carboxyfluorescein succinimidyl ester (CFSE) is an effective and popular means to monitor lymphocyte division1-3.  CFSE covalently labels long-lived ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

세균성 접착 연구에 전단 응력을 소개합니다

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Antigens Protected Functional Red Blood Cells By The Membrane Grafting Of Compact Hyperbranched Polyglycerols

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell anemia 1-3. Due to the presence of multitude of antigens on the RBC surface (~308 known antigens 4), patients in the chronic blood transfusion therapy develop alloantibodies due to the miss match of minor antigens on transfused RBCs 4, 5. Grafting of hydrophilic polymers such as polyethylene glycol (PEG) and hyperbranched polyglycerol (HPG) forms an exclusion layer on RBC membrane that prevents the interaction of antibodies with surface antigens without affecting the passage of small molecules such as oxygen ,glucose, and ions3. At present no method is available for the generation of universal red blood donor cells in part because of the daunting challenge presented by the presence of large number of antigens (protein and carbohydrate based) on the RBC surface and the development of such methods will significantly improve transfusion safety, and dramatically improve the availability and use of RBCs. In this report, the experiments that are used to develop antigen protected functional RBCs by the membrane grafting of HPG and their characterization are presented. HPGs are highly biocompatible compact polymers 6, 7, and are expected to be located within the cell glycocalyx that surrounds the lipid membrane 8, 9 and mask RBC surface antigens10, 11.

The cell membrane modification of red blood cells (RBCs) with hyperbranched polyglycerol (HPG) is presented. Modified RBCs were characterized by aqueous two phase partitioning, osmotic fragility and complement mediated lysis. The camouflage of surface proteins and antigens was evaluated using the flow cytometry and Micro Typing System (MTS) blood phenotyping cards.

항원은 컴팩트 Hyperbranched Polyglycerols 중 이식 막으로 기능 적혈구를 보호

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell ...

Methodology for the Efficient Generation of Fluorescently Tagged Vaccinia Virus Proteins

Tagging of viral proteins with fluorescent proteins has proven an indispensable approach to furthering our understanding of virus-host interactions. Vaccinia virus (VACV), the live vaccine used in the eradication of smallpox, is particularly amenable to fluorescent live-cell microscopy owing to its large virion size and the ease with which it can be engineered at the genome level. We report here an optimized protocol for generating recombinant viruses. The minimal requirements for targeted homologous recombination during vaccinia replication were determined, which allows the simplification of construct generation. This enabled the alliance of transient dominant selection (TDS) with a fluorescent reporter and metabolic selection to provide a rapid and modular approach to fluorescently label viral proteins. By streamlining the generation of fluorescent recombinant viruses, we are able to facilitate downstream applications such as advanced imaging analysis of many aspects of the virus-host interplay that occurs during virus replication.

A rapid and modular protocol for the generation of recombinant vaccinia viruses expressing fluorescently&#160;tagged proteins simultaneously using the method of transient dominant selection is described here.

형광 태그 바시니아 바이러스 단백질의 효율적인 생성을위한 방법론

Tagging of viral proteins with fluorescent  proteins has proven an indispensable approach to furthering our understanding  of virus-host interactions. ...

Assay of Adhesion Under Shear Stress for the Study of T Lymphocyte-Adhesion Molecule Interactions

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and interaction with antigen presenting cells (APC) in the formation of immunological synapses. These two contexts of T cell adhesion differ in that T cell-APC interactions can be considered static, while T cell-blood vessel interactions are challenged by the shear stress generated by circulation itself. T cell-APC interactions are classified as static in that the two cellular partners are static relative to each other. Usually, this interaction occurs within the lymph nodes. As a T cell interacts with the blood vessel wall, the cells arrest and must resist the generated shear stress.1,2 These differences highlight the need to better understand static adhesion and adhesion under flow conditions as two distinct regulatory processes. The regulation of T cell adhesion can be most succinctly described as controlling the affinity state of integrin molecules expressed on the cell surface, and thereby regulating the interaction of integrins with the adhesion molecule ligands expressed on the surface of the interacting cell. Our current understanding of the regulation of integrin affinity states comes from often simplistic in vitro model systems. The assay of adhesion using flow conditions described here allows for the visualization and accurate quantification of T cell-epithelial cell interactions in real time following a stimulus. An adhesion under flow assay can be applied to studies of adhesion signaling within T cells following treatment with inhibitory or stimulatory substances. Additionally, this assay can be expanded beyond T cell signaling to any adhesive leukocyte population and any integrin-adhesion molecule pair.

This flow adhesion assay provides a simple, high impact model of T cell-epithelial cell interactions. A syringe pump is used to generate shear stress, and confocal microscopy captures images for quantification. The goal of these studies is to effectively quantify T cell adhesion using flow conditions.

T 림프구 - 접착 분자 상호 작용의 연구에 대한 접착에서 전단 응력의 분석

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and ...

Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry

Trypanosoma brucei, a protozoan parasite that causes both Human and Animal African Trypanosomiasis (known as sleeping sickness and nagana, respectively) cycles between a tsetse vector and a mammalian host. It evades the mammalian host immune system by periodically switching the dense, variant surface glycoprotein (VSG) that covers its surface. The detection of antigenic variation in Trypanosoma brucei can be both cumbersome and labor intensive. Here, we present a method for quantifying the number of parasites that have 'switched' to express a new VSG in a given population. The parasites are first stained with an antibody against the starting VSG, and then stained with a secondary antibody attached to a magnetic bead. Parasites expressing the starting VSG are then separated from the rest of the population by running the parasites over a column attached to a magnet. Parasites expressing the dominant, starting VSG are retained on the column, while the flow-through contains parasites that express a new VSG as well as some contaminants expressing the starting VSG. This flow-through population is stained again with a fluorescently labeled antibody against the starting VSG to label contaminants, and propidium iodide (PI), which labels dead cells. A known number of absolute counting beads that are visible by flow cytometry are added to the flow-through population. The ratio of beads to number of cells collected can then be used to extrapolate the number of cells in the entire sample. Flow cytometry is used to quantify the population of switchers by counting the number of PI negative cells that do not stain positively for the starting, dominant VSG. The proportion of switchers in the population can then be calculated using the flow cytometry data.

Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry

African trypanosomes grown in vitro undergo antigenic variation at a low rate, such that populations are made up of parasites expressing a dominant variant surface glycoprotein (VSG) type and a small population of "switched" variants. This protocol describes a fast method for detecting and quantifying these populations.

검출<em&gt; 트리파노소마 (Trypanosoma) brucei</em자기 활성 셀 정렬 및 유동 세포 계측법에 의해&gt; 변형 표면 당 단백질 전환

Trypanosoma brucei, a protozoan parasite that causes both Human and Animal African Trypanosomiasis (known as sleeping sickness and nagana, respectively) ...

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.

Use of a Monocyte Monolayer Assay to Evaluate Fc&amp;#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 ...