나는 그림 1의 데이터를 제공 Sreemanti Basu 감사하고 싶습니다. 이 작품은 NIH 부여 AI069358 및 BloodCenter 연구 재단에 의해 부분적으로 지원되었다.

A. 래빗 보완 <ol><li> 주식 토끼 보완은 상온에서 해동 후 0.5 및 / 또는 1 ML 볼륨에 aliquoted과 ° C 장기 저장을위한 -20-80에서 냉동해야합니다. </li><li> 보완 각각의 많은 최적의 작업 농도를 결정하기 위해 titrated해야합니다. 일반적으로, 1시 10분에서 1시 20분까지의 희석이 최적입니다. </li><li> 토끼 보완은 항상 세포의 최종 사용에 따라 이전에 판매 presterilized 아니라, 보완은 aliquoted 때 소독 필터링 사전 세포 이외에 냉동하거나해야합니다. </li></ol> B. 항체 선택 <ol><li> 모든 항체 따라서 항체가 처음 활동에 대한 검사를해야하며, 보완 활성화에 효과적입니다. </li><li> 마우스 및 쥐 mAb 모두 보완 활성화에 효과적입니다. </li><li> IgM 항체는 IgG 다음 보완 유도된 세포 용해에 사용하기 위해 일반적으로 가장 효율적인 수 있습니다. 최고의 보완 활성화에 대한 IgG의 isotypes, 종족 간의 다릅니다. </li><li> 각 항체는 10 7 셀 / ML의 세포 밀도에 최적 사용하기 위해 titrated해야합니다. </li></ol> C. 기본 프로토콜 <ol><li> 5 시작 세포 인구를 조절 매체를 포함하는 열 inactivated FCS에서 X 10 7 세포 / ML. 세포의 최종 볼륨을 두 번 시작 볼륨 것입니다. 고갈을 계량 수 있도록 몇 가지 세포를 저장하는 기억하십시오. </li><li> 각 항체는 적절한 정해진 농도 또는 희석에 세포 현탁액에 추가되어 있어야합니다. 잘 역전하여 세포를 섞는다. </li><li> 필요한 보완의 양을 계산합니다. 상온에서 보완 주식을 녹여. 보완이 멸균되지 않은 경우, 그것이 낮은 단백질 바인딩 주사기 팁 필터를 사용하여 소독. 주사기하려면, 보완을 추가하기 전에 중간 여러 ML을 추가하고 세포와 항체가 들어있는 튜브에 직접 필터. </li><li> 세포는 1 X 10 7 셀 / ML에 있습니다 있도록 튜브의 마지막 볼륨을 조정합니다. </li><li> 15 분 간격으로 혼합 한 시간 동안 37 ° C에서 세포를 품어. </li><li> 세포를 원심과 뜨는 폐기하십시오. </li><li> 매체와 함께 두 번 씻으십시오. 세포 파편 폴리스티렌 튜브의 측면에 집중합니다. 죽은 세포와 부스러기는 셀 스트레이너 또는 밀도 기울기 원심 분리에 의해 제품을 사용하여 제거하실 수 있습니다. </li><li> 고갈 전후 관심의 세포 인구를 비교하여 세포 순결을 확인합니다. 유동세포계측법 및 immunohistochemistry는 세포 순도를 결정하는 두 가지 좋은 방법입니다. </li></ol> 대표 결과 한 라운드 후 보완, 세포 고갈을 활성화에 매우 효율적입니다 mAb를 사용하면하면 다음 95% 큽니다. 이것은 우리가 유동세포계측법에 의해 TCRβ 표현 (eBioscience, 샌디에고, CA)에 의해 감지와 같은 32.2 %의 αβ의 T 세포로 구성된 마우스 splenocytes 시작 그림 1에 표시됩니다. 우리는 mAb Thy1에 대한 구체적인 모든 T 세포에 의해 표현 단백질지만, 다른 건 못 leukocytes를 사용하여 T 세포를 고갈. 우리가 사용하는 항체는 Y - 19 쥐를 IgG2c이되었습니다. 설명된 절차에 따라, TCRβ 인구는 1.56 % 이상 95 % 감소로 감소했다. <img src="/files/ftp_upload/1487/1487fig1.jpg" alt="그림 1" /> 그림 1. thy1의 고갈은 + 보완 활성화하여 지라 T 세포. 총 마우스 splenocytes는 RBC의 비장과 용해의 균질하여 얻을 수 있었다. 안티 thy1하고 사전에 정해진 농도의 아기 토끼 보완은 최종 세포 농도는 1 X 10 7 셀 / ML 것을 같은 splenocytes의 단일 세포 현탁액에 추가되었습니다. 37에서 1 시간 동안 배양 후 ° C, 세포가 두 번 씻어 및 세포 생존을위한 trypan 파랑 배제에 의해 평가되었다. 고갈의 효율 고갈 (왼쪽 패널) 전에 고갈 (오른쪽 패널) 이후 TCRβ + 세포의 비율을 비교 유동세포계측법에 의해 평가되었다. 수평 막대는 긍정적인 얼룩을 나타내며 막대 위에있는 숫자는 긍정 %입니다.

<ol>
	<li>Janeway, C., Travers, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Immune+System+in+Health+and+Disease.">The Immune System in Health and Disease.</a> Immunobiology. , (1994).</li><li>Jones, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+activities+of+antibodies+against+brain-associated+T+cell+antigens+II.+Stimulation+of+T+cell-induced+B+cell+proliferation.">Functional activities of antibodies against brain-associated T cell antigens II. Stimulation of T cell-induced B cell proliferation.</a> Eur J Immunol. 12, 30-37 (1982).</li></ol>

세포 인구의 정화는 기능의 연구에 필요한 일반적인 절차입니다. 여기 우리는 항원 - 항체 및 구체적인 보완 고갈을 사용하여 셀 인구 파괴의 신속하고 효과적인 방법을 설명합니다. 보완 - 활성화 항체가 혈통 특정 마커를 사용할 경우 모든 세포 인구의 고갈이 형성될 수 있습니다. 여기에 우리가 Thy1에 대한 mAb를 사용하여 특정 T 세포의 고갈을 시연, 단백질은 본질적으로 모든 T 세포 집단에 의해 표시됩니다. 우리는 αβ T 세포의 95 % 이상 고갈 나머지 splenocytes는 68%에서 98 %로 증가했다. 세포 순결의이 수준은 외과와 ​​자석 구슬 분리 등 다른 정화 전략과 비슷합니다. 보완 고갈이 시작 셀 인구가 획득되면 1.5 시간에 수행할 수있는 빠른 방법입니다. 보완 고갈의 또 다른 장점은 고가의 장비와 시약을 필요로하지 않기 때문에 모든 실험실이 기법을 수행할 수있다는 것입니다. 필요한 장비의 유일한 주요 작품은 물 목욕과 원심 분리기입니다. 토끼 보완은 저렴한 비용으로 다양한 공급 업체에서 구입하실 수 있습니다 (시약의 표 참조) 많은 mAb는 ATTC에서 구입하여 비용을 관리하는 로컬 성장하실 수 있습니다. 반면, 외과는 여섯 숫자와 비싼 찬란 태그 mAb의 비용 셀 정렬 용량 흐름 cytometer이 필요합니다. 마그네틱 비드 분리 알맞게 가격이지만, 치명타가 될 수 있습니다 자석과 자석 구슬의 구입이 필요합니다. 또한, 일부 자기 비드 분리 전략은 또한 컬럼의 구입이 필요합니다. 세포 고갈에 대한 보완의 사용에 가장 큰 걸림돌이 필요한 항원 특이성과 mAb를 활성화 보완의 가용성이다. 인간, 마우스 및 쥐 항체가 모두 보완 문제를 해결하는 능력을 가지고 있지만, 일부 isotypes 다른보다됩니다. 세 종 IgM은 보완을 고치에서 매우 효과이지만, IgG의 isotypes이 다릅니다. 따라서 모든 mAb 먼저 그 효과를 확인하기 위해 파일럿 연구에서 테스트해야합니다. 고효율되지 않은 항체 들어, 보완 고갈의 두 라운드가 남아있는 세포 집단의 생존에 영향을주지 않고 수행할 수 있습니다. 비용 절감으로 항체는 최적의 효과에 대한 titrated해야합니다. 또한, 사용되는 항체는 정화가 필요가 없습니다. 정화 어렵습니다 IgM 항체, 특히 편리합니다 마찬가지로 효과적으로 하이 브리 도마 supernatants 및 복수 유체 작업. 항체와 마찬가지로 그것은 보완이 파일럿 연구에서 titrated하는 것이 필수적입니다. 농도의 너무 높은가 아닌 타겟 세포 인구의 생존 능력의 손실을 초래합니다. 또한 중요 부화 시간이 60 분 초과하지 않는다는 것입니다. 우리의 프로토콜에서는, 우리는 함께 항체와 보완을 공동 품어. 그러나, 세포는 15-30분에 대한 항체로 분류 사전 보완을 추가로 씻어 수 있습니다. 이 전략을 사용하는 경우와 같은 패치 및 상한 또는 수용체 국제화에 의해 일부 단백질과 발생, 세포 표면에있는 세포 항원의 손실을 방지하기 위해 얼음에 항체 incubations을 수행할 수 있는지 확인하십시오. 고효율이지만, 다른 방법에 비해 소모를 보완하기 위해 주요 단점은 그것이 긍정적인 선택에 사용할 수 없다는 것입니다. 그러므로 그것은 세포의 subpopulations를 얻을 어렵습니다. 예를 들어, 최고의 외과에 의해 수행 될 follicular의 B 세포에서 한계 영역 B 세포를 정화하기 어려운 것입니다. 이 상황에서, 보완 정품 인증이 정렬되는 세포의 수를 줄이기 위해 T 세포를 고갈하는 데 사용할 수 있습니다. 이 전략은 비용을 절감하는 것이있는, 정렬 시간을 줄일 것이며, 세포에 덜 거칠 것입니다. 특정 세포 집단의 정화는 일반적인 요구 사항이기 때문에 전략의 번호가 개발되었습니다. 보완 고갈 전략의 주요 장점은 경제성, 기술적 단순 및 시간 절감하고 있습니다.

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		<thead>
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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>3-4 week baby rabbit complement</td>
<td>&nbsp;</td>
<td>Pel-Freez Biologicals</td>
<td>31061-3</td>
<td>&nbsp;</td>
<tr>
<td>Water bath</td>
<td>&nbsp;</td>
<td>various</td>
<td>various </td>
<td>An incubator set at 37 &deg;C can also be used.</td>
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depletion of specific cell populations by complement depletion

면역 세포 집단의 정화는 종종 자신의 고유의 기능을 연구하기 위해 필요합니다. 이러한 실시간 PCR과 microarray 분석 등 특히, 분자 접근법에서 고순도로 세포 인구의 격리를 필요로합니다. 일반적으로 사용되는 정화 전략은 형광 활성 세포 분류 (외과), 마그네틱 비드 분리 및 보완 고갈을 포함합니다. 세 전략, 보완의 고갈은 빠르고 저렴, 세포가 높은 세포 수율에 부드러운되는 장점을 제공합니다. 보완 시스템이 활성화되면 세포 사망의 결과 세포 표면에 기공을 형성하는 멤브레인 - 공격 복잡한 형성에 culminating proteolytic 층계를 시작 플라즈마 단백질의 다수로 구성되어 있습니다<sup&gt; 1</sup&gt;. 고전 경로는 IgM과 IgG 항체에 의해 활성화되며 첫 살해 박테리아를위한 메커니즘으로 설명했다. 단클론 항체 (mAb)의 세대, 보완 층계는 항원에 특정한 방식으로 모든 세포 인구를 lyse하는 데 사용할 수 있습니다. 보완 층계에 의해 세포의 고갈은 보완의 고정 항원에 특정한 항체를 시작 셀 인구 토끼 보완의 추가에 의해 이루어진다. 세포 ° C와 lysed 세포 세척 후 두 라운드에 의해 제거 37 한 시간 동안 incubated 수 있습니다. 보완의 고정을위한 고효율 MAb은 일반적으로 타겟 세포 인구의 95-100%를 고갈. 대상 세포 인구를위한 정화 전략에 따라 보완 고갈은 세포 정화 또는 다음 추가 후속 방법으로 정화 수있는 세포 집단의 강화에 사용될 수 있습니다.

Watch this Scientific Journal Video about Depletion of Specific Cell Populations by Complement Depletion at JoVE.com

Depletion of Specific Cell Populations by Complement Depletion

효과적으로 정화가 자주 필요한 면역 세포 집단의 기능을 연구합니다. 보완 고갈은 고순도와 면역 세포 인구의 격리를위한 신속하고 저렴한 방법입니다.

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 ...

depletion-of-specific-cell-populations-by-complement-depletion

Research

JoVE Journal

Immunology and Infection

22.0K 조회수.  Blood Research Institute. 효과적으로 정화가 자주 필요한 면역 세포 집단의 기능을 연구합니다. 보완 고갈은 고순도와 면역 세포 인구의 격리를위한 신속하고 저렴한 방법입니다.

비디오: Depletion of Specific Cell Populations by Complement Depletion

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 ...

연구

면역학 및 감염병학

Measuring the 50% Haemolytic Complement (CH50) Activity of Serum

The complement system is a group of proteins that when activated lead to target cell lysis and facilitates phagocytosis through opsonisation. Individual complement components can be quantified however this does not provide any information as to the activity of the pathway. The CH50 is a screening assay for the activation of the classical complement pathway (Fig 1) and it is sensitive to the reduction, absence and/or inactivity of any component of the pathway. The CH50 tests the functional capability of serum complement components of the classical pathway to lyse sheep red blood cells (SRBC) pre-coated with rabbit anti-sheep red blood cell antibody (haemolysin). When antibody-coated SRBC are incubated with test serum, the classical pathway of complement is activated and haemolysis results. If a complement component is absent, the CH50 level will be zero; if one or more components of the classical pathway are decreased, the CH50 will be decreased. A fixed volume of optimally sensitised SRBC is added to each serum dilution. After incubation, the mixture is centrifuged and the degree of haemolysis is quantified by measuring the absorbance of the haemoglobin released into the supernatant at 540nm. The amount of complement activity is determined by examining the capacity of various dilutions of test serum to lyse antibody coated SRBC. This video outlines the experimental steps involved in analysing the level of complement activity of the classical complement pathway.

Measuring the 50% Haemolytic Complement CH50 Activity of Serum

The classical pathway is activated by antibody and culminates in target cell lysis. The CH50 assay provides a measure of the complement activity of a serum sample. This video demonstrates the steps involved in determining the CH50 of a serum sample, the calculations and interpreting of results.

50 % Haemolytic 보완 (CH 측정 50</sub혈청>) 활동

The complement system is a group of proteins that when activated lead to target cell lysis and facilitates phagocytosis through opsonisation. Individual ...

Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the generation of extreme population diversity that facilitates virus adaptation and evolution. Increasing evidence shows that the intrinsic error rates, and the resulting mutation frequencies, of RNA viruses can be modulated by subtle amino acid changes to the viral polymerase. Although biochemical assays exist for some viral RNA polymerases that permit quantitative measure of incorporation fidelity, here we describe a simple method of measuring mutation frequencies of RNA viruses that has proven to be as accurate as biochemical approaches in identifying fidelity altering mutations. The approach uses conventional virological and sequencing techniques that can be performed in most biology laboratories. Based on our experience with a number of different viruses, we have identified the key steps that must be optimized to increase the likelihood of isolating fidelity variants and generating data of statistical significance. The isolation and characterization of fidelity altering mutations can provide new insights into polymerase structure and function1-3. Furthermore, these fidelity variants can be useful tools in characterizing mechanisms of virus adaptation and evolution4-7.

The present article describes the steps required to isolate and characterize RNA polymerase fidelity variants of RNA viruses and how to use mutation frequency data to confirm fidelity changes in tissue culture.

바이러스 변이 주파수의 RNA 바이러스 및 특성화의 피델리티 변종의 분리

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the ...

Depletion and Reconstitution of Macrophages in Mice

Macrophages are critical players in the innate immune response to infectious challenge or injury, initiating the innate immune response and directing the acquired immune response. Macrophage dysfunction can lead to an inability to mount an appropriate immune response and as such, has been implicated in many disease processes, including inflammatory bowel diseases. Macrophages display polarized phenotypes that are broadly divided into two categories. Classically activated macrophages, activated by stimulation with IFN&gamma; or LPS, play an essential role in response to bacterial challenge whereas alternatively activated macrophages, activated by IL-4 or IL-13, participate in debris scavenging and tissue remodeling and have been implicated in the resolution phase of inflammation. During an inflammatory response in vivo, macrophages are found amid a complex mixture of infiltrating immune cells and may participate by exacerbating or resolving inflammation. To define the role of macrophages in situ in a whole animal model, it is necessary to examine the effect of depleting macrophages from the complex environment. To ask questions about the role of macrophage phenotype in situ, phenotypically defined polarized macrophages can be derived ex vivo, from bone marrow aspirates and added back to mice, with or without prior depletion of macrophages. In the protocol presented here clodronate-containing liposomes, versus PBS injected controls, were used to deplete colonic macrophages during dextran sodium sulfate (DSS)-induced colitis in mice. In addition, polarized macrophages were derived ex vivo and transferred to mice by intravenous injection. A caveat to this approach is that clodronate-containing liposomes deplete all professional phagocytes, including both dendritic cells and macrophages so to ensure the effect observed by depletion is macrophage-specific, reconstitution of phenotype by adoptive transfer of macrophages is necessary. Systemic macrophage depletion in mice can also be achieved by backcrossing mice onto a CD11b-DTR background, which is an excellent complementary approach. The advantage of clodronate-containing liposome-mediated depletion is that it does not require the time and expense involved in backcrossing mice and it can be used in mice regardless of the background of the mice (C57BL/6, BALB/c, or mixed background).

Macrophages play a central role in homeostasis and pathology in many tissues. The protocol presented here describes methods for depleting macrophages in vivo, deriving polarized macrophages from bone marrow aspirates, and adoptively transferring macrophages into mice. These techniques allow determination of the role that polarized macrophages play in health and disease.

마우스의 Macrophages의 고갈과 Reconstitution

Macrophages are critical players in the innate  immune response to infectious challenge or injury, initiating the innate immune  response and directing ...

Visualization of the Immunological Synapse by Dual Color Time-gated Stimulated Emission Depletion (STED) Nanoscopy

Natural killer cells form tightly regulated, finely tuned immunological synapses (IS) in order to lyse virally infected or tumorigenic cells. Dynamic actin reorganization is critical to the function of NK cells and the formation of the IS. Imaging of F-actin at the synapse has traditionally utilized confocal microscopy, however the diffraction limit of light restricts resolution of fluorescence microscopy, including confocal, to approximately 200 nm. Recent advances in imaging technology have enabled the development of subdiffraction limited super-resolution imaging. In order to visualize F-actin architecture at the IS we recapitulate the NK cell cytotoxic synapse by adhering NK cells to activating receptor on glass. We then image proteins of interest using two-color stimulated emission depletion microscopy (STED). This results in &#60;80 nm resolution at the synapse. Herein we describe the steps of sample preparation and the acquisition of images using dual color STED nanoscopy to visualize F-actin at the NK IS. We also illustrate optimization of sample acquisition using Leica SP8 software and time-gated STED. Finally, we utilize Huygens software for post-processing deconvolution of images.

Visualization of the Immunological Synapse by Dual Color Time-gated Stimulated Emission Depletion STED Nanoscopy

Here we illustrate the protocol for imaging by two-color STED nanoscopy the cytotoxic immune synapse of NK cells recapitulated on glass. Using this method we obtain sub-100 nm resolution of synapse proteins and the cytoskeleton.

이중 색깔 시간 게이트 자극 방출 고갈 (STED) Nanoscopy에 의한 면역 시냅스의 시각화

Natural killer cells form tightly regulated, finely tuned immunological synapses (IS) in order to lyse virally infected or tumorigenic cells. Dynamic ...

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 ...

Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied by adverse side effects such as a reduced ability to control infections or malignancies. Therefore, new approaches need to be developed that target only the disease mediating cells and leave the remaining immune system intact. Over the past decade a variety of cell based immunotherapy strategies to modulate T cell mediated immune responses have been developed. Most of these approaches rely on tolerance-inducing antigen presenting cells (APC). However, in addition to being technically difficult and cumbersome, such cell-based approaches are highly sensitive to cytotoxic T cell responses, which limits their therapeutic capacity. Here we present a protocol for the generation of non-cellular killer artificial antigen presenting cells (KaAPC), which allows for the depletion of pathologic T cells while leaving the remaining immune system untouched and functional. KaAPC is an alternative solution to cellular immunotherapy which has potential for treating autoimmune diseases and allograft rejections by regulating undesirable T cell responses in an antigen specific fashion.

Killer Artificial Antigen Presenting Cells KaAPC for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Guidelines are presented for the generation of killer artificial antigen presenting cells, KaAPC, an efficient tool for in vitro depletion of human antigen-specific T cells and an alternative solution to cellular immunotherapy for the treatment of T cell-mediated autoimmune diseases in an antigen-specific fashion without compromising the remaining immune system.

효율적인위한 킬러 인공 항원 제시 세포 (KaAPC)<em&gt; 체외</em&gt; 인간의 항원 특이 T 세포의 고갈

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied ...

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are able to replicate in the deep lung. Phagocytosis of Cryptococcus by macrophages is one of the ways that the disease is able to spread into the central nervous system to cause lethal meningoencephalitis. Therefore, study of the association between Cryptococcus and macrophages is important to understanding the progression of the infection. The present study describes a step-by-step protocol to study macrophage infectivity by C. neoformansin vitro. Using this protocol, the role of host sterols on host-pathogen interactions is studied. Different concentrations of methyl--cyclodextrin (MCD) were used to deplete cholesterol from murine reticulum sarcoma macrophage-like cell line J774A.1. Cholesterol depletion was confirmed and quantified using both a commercially available cholesterol quantification kit and thin layer chromatography. Cholesterol depleted cells were activated using Lipopolysacharide (LPS) and Interferon gamma (IFN&#947;) and infected with antibody-opsonized Cryptococcus neoformans wild-type H99 cells at an effector-to-target ratio of 1:1. Infected cells were monitored after 2 hr of incubation with C. neoformans and their phagocytic index was calculated. Cholesterol depletion resulted in a significant reduction in the phagocytic index. The presented protocols offer a convenient method to mimic the initiation of the infection process in a laboratory environment and study the role of host lipid composition on infectivity.

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

In this article, a protocol for infection of macrophages with Cryptococcus neoformans is described. Also, a method for sterol depletion from the macrophages is explained. These protocols provide a guide to study fungal infections in vitro and examine the role of sterols in such infections.

대 식세포 콜레스테롤 고갈과의 식균 작용에 미치는 영향<em&gt; 크립토 콕 쿠스 네오 포르</em

Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are ...

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, cloning TCRs specific for various tumor-associated antigens has been the goal of many studies. To elicit an effective T cell response, the TCR must recognize the target antigen with optimal affinity. However, cloning such TCRs has been a challenge and many available TCRs possess sub-optimal affinity for the cognate antigen. In this protocol, we describe a method of cloning de novo high affinity antigen-specific TCRs using existing TCRs by exploiting hemichain centricity. It is known that for some TCRs, each TCR&#945; or TCR&#946; hemichain do not contribute equally to antigen recognition, and the dominant hemichain is referred to as the centric hemichain. We have shown that by pairing the centric hemichain with counter-chains differing from the original counter-chain, we are able to maintain the antigen specificity, while modulating its interaction strength for the cognate antigen. Thus, the therapeutic potential of a given TCR can be improved by optimizing the pairing between the centric and counter hemichains.

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

Herein we describe a novel method to generate antigen-specific T cell receptors (TCRs) by pairing the TCR&#945; or TCR&#946; of an existing TCR, possessing the antigen-specificity of interest, with complementary hemichain of the peripheral T cell receptor repertoire. The de novo generated TCRs retain antigen-specificity with varying affinity.

생성<em&gt; 드 노보</em&gt; 중심 Hemichain의 레트로 바이러스 형질 도입에 의해 항원 특이 적 인간 T 세포 수용체

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, ...

Visualizing the Actin and Microtubule Cytoskeletons at the B-cell Immune Synapse Using Stimulated Emission Depletion (STED) Microscopy

B cells that bind to membrane-bound antigens (e.g., on the surface of an antigen-presenting cell) form an immune synapse, a specialized cellular structure that optimizes B-cell receptor (BCR) signaling and BCR-mediated antigen acquisition. Both the remodeling of the actin cytoskeleton and the reorientation of the microtubule network towards the antigen contact site are essential for immune synapse formation. Remodeling of the actin cytoskeleton into a dense peripheral ring of F-actin is accompanied by polarization of the microtubule-organizing center towards the immune synapse. Microtubule plus-end binding proteins, as well as cortical plus-end capture proteins mediate physical interactions between the actin and microtubule cytoskeletons, which allow them to be reorganized in a coordinated manner. Elucidating the mechanisms that control this cytoskeletal reorganization, as well as understanding how these cytoskeletal structures shape immune synapse formation and BCR signaling, can provide new insights into B cell activation. This has been aided by the development of super-resolution microscopy approaches that reveal new details of cytoskeletal network organization. We describe here a method for using stimulated emission depletion (STED) microscopy to simultaneously image actin structures, microtubules, and transfected GFP-tagged microtubule plus-end binding proteins in B cells. To model the early events in immune synapse formation, we allow B cells to spread on coverslips coated with anti-immunoglobulin (anti-Ig) antibodies, which initiate BCR signaling and cytoskeleton remodeling. We provide step-by-step protocols for expressing GFP fusion proteins in A20 B-lymphoma cells, for anti-Ig-induced cell spreading, and for subsequent cell fixation, Immunostaining, image acquisition, and image deconvolution steps. The high-resolution images obtained using these procedures allow one to simultaneously visualize actin structures, microtubules, and the microtubule plus-end binding proteins that may link these two cytoskeletal networks.

Visualizing the Actin and Microtubule Cytoskeletons at the B-cell Immune Synapse Using Stimulated Emission Depletion STED Microscopy

We present a protocol for using STED microscopy to simultaneously image actin structures, microtubules, and microtubule plus-end binding proteins in B cells that have spread on coverslips coated with antibodies to the B-cell receptor, a model for the initial phase of immune synapse formation.

말라와 자극된 방출 소모 (호텔 위치) 현미경을 사용 하 여 B 세포 면역 냅에 Microtubule Cytoskeletons 시각화

B cells that bind to membrane-bound antigens (e.g., on the surface of an antigen-presenting cell) form an immune synapse, a specialized cellular structure ...