저자는 촬영에 대한 기술과 미스터 폴 목사는 수행 미스 로라 매튜스를 감사하고 싶습니다. 이 프로젝트는 사우스 오스트 레일 리아 대학, 연구소의 의학 프로그램에 의해 투자되었다.

5 배 Veronal 버퍼 살린 (VBS)의 준비 <ol><li> Veronal 버퍼 살린 (VBS)를 준비하려면, 별도의 세 가지 솔루션을 준비해야합니다. </li><li> 증류수의 350ml에 나트륨 Barbitone의 NaCl과 0.94gm의 21.25gm을 용해하여 용액 1을 준비합니다. NaCl과 나트륨 Barbitone의 최종 농도는 각각 1.02M과 13mM 있습니다. </li><li> 뜨거운 증류수의 125ml에 Barbitone의 1.44gm을 용해하여 용액 2를 준비합니다. Barbitone의 최종 농도는 62.5mM입니다. </li><li> 증류수의 100ml에 CaCl 2 MgCl 2 4.41gm의 20.33gm을 용해하여 용액 세를 준비합니다. MgCl 2, CaCl 2의 최종 농도는 각각 2.18M과 440mM입니다. </li><li> 믹스 솔루션 1, 2 룸 온도 냉각. </li><li> 통합 솔루션이 냉각되면, 솔루션 3 1.25ml를 추가하고 1M HCL을 사용하여 7.3-7.5 수있는 산도를 조정합니다. </li><li> 배 재고 솔루션을 준비하는 증류수로 500ml로 최종 볼륨을 조정합니다. </li><li> 1X 작업 솔루션을 준비하려면, 증류수와 주식 1:5 희석. </li></ol> haemolysin와 양 적혈구의 Sensitisation <ol><li> 첫째 VBS로 1시 50분를 diluting하여 haemolysin 준비 </li><li> SRBC의 위해 4ml는 VBS의 6ml를 추가하고 부드럽게 반전에 의해 믹스 </li><li> 600g X 오분에서 원심 분리기 </li><li> 뜨는을 취소하고 세포를 다른 2 번 씻어 </li><li> 최종 세척 후, 세포를 싣고 900g X의 오분의 세포를 원심 분리기 </li><li> 뜨는을 취소하고 포장 세포의 10 % 즉, 솔루션 0.5ml를 준비하는 충분한 VBS에있는 세포를 resuspend는 버퍼의 5 ML에 resuspended 아르 </li><li> 지속적으로 소용돌이 치는 동안 세포 haemolysin의 동일한 음량을 (토끼 안티 양 적혈구 세포 항체) 추가 Dropwise </li><li> 물 목욕 30 분 30 ° C에서 알을 품다 </li><li> 부드럽게 세포 매 십오분 믹스 </li><li> Sensitised SRBC 4에서 하룻밤 저장할 수 있습니다 ° C </li></ol> CH 50 검정 <ol><li> 1시 8분, 1시 16분, 1시 32분, 1:64 및 1:128와 중복의 튜브 일련의 라벨. </li><li> 중복 각 VBS의 제어 및 시험 혈청의 두 배 시리얼 dilutions 일련의 준비 </li><li> 1시 4분 (100ml + 300ml 혈청 VBS)에서 시작하고 다음 표시된 튜브에 시료의 200ml를 전송할 수 있습니다. </li><li> 신선한 피펫 팁과 다음 희석에 dilutions 및 전송 200ml 사이에 철저히 섞는다. </li><li> 다섯 dilutions이 이루어질 때까지 반복합니다. </li><li> 최종 1:128 희석에서 200ml를 폐기하십시오. </li><li> 모든 튜브에 정지 sensitized SRBC의 200ml를 추가합니다. </li><li> 라벨 두 개의 튜브로 빈과 sensitized SRBC + 200ml VBS의 200ml를 추가합니다. 이러한 튜브는 VBS의 SRBC의 자발적인 용해를 측정합니다. </li><li> TOTAL 용해와 같은 다른 두 개의 튜브 라벨과 sensitized SRBC + 200ml 증류수의 200ml 추가합니다. </li><li> 부드럽게 모든 튜브를 섞는다. </li><li> 37 ° C 십오분 후 혼합 waterbath 30 분 알을 품다. </li><li> 퇴적물 RBCs를 5 분 1천5백g에서 샘플을 원심 분리기. </li><li> 96 잘 플랫 바텀 플레이트에 잘 각 튜브의 표면에 뜨는의 100ml를 전송합니다. </li><li> 각 잘하는 증류수의 100ml를 추가합니다. </li><li> 플레이트 분광 광도계를 사용하여 540nm에서 샘플 흡광도를 읽어보십시오. </li></ol> 계산 <ol><li> 각 샘플에 대한 평균 흡광도를 계산 </li><li> 모든 샘플에서 빈 흡광도 (자발적인 용해)을 빼기 </li><li> 다음 수식을 사용하여 각 희석에 대한 %의 용해를 계산 : <img src="/files/ftp_upload/1923/1923eq1.jpg" alt="방정식" /></li><li> 수평축에 혈청 희석 대 비율 용해 (수직 축)를 플롯. </li><li> 제어 및 시험 혈청 (그림 2) 50 % haemolysis에 필요한 희석을 계산합니다. </li></ol> 대표 결과 : 비디오는 대표적인 결과의 예를 포함하고 있습니다. 연습에서는 컨트롤 혈청 또한 시험 혈청 동시에 실행하고 같은 방식으로 처리됩니다. 아래 (표 1) 검사 혈청 샘플에서 예제 데이터입니다. 데이터는 5.3에서 제시한 공식에 따라 조작할 수 있습니다. <table border="1"><tbody><tr><td> 견본 </td><td> OD 540 </td><td> 평균 </td><td></td><td></td></tr><tr><td> 공백 </td><td> 0.042, 0.044 </td><td> 0.043 </td><td></td><td></td></tr><tr><td> 총 용해 </td><td> 0.183, 0.183 </td><td> 0.183 </td><td></td><td></td></tr><tr><td colspan="5"></td></tr><tr><td> 노동 희석 </td><td> OD 540 </td><td> 평균 </td><td> 평균 - 빈 </td><td> %의 용해 </td></tr><tr><td> 1시 8분 </td><td> 0.200, 0.219 </td><td> 0.210 </td><td> 0.168 </td><td> 116 </td></tr><tr><td> 1시 16분 </td><td> 0.179, 0.173 </td><td> 0.176 </td><td> 0.134 </td><td> 93 </td></tr><tr><td> 1시 32분 </td><td> 0.134, 0.110 </td><td> 0.122 </td><td> 0.08 </td><td> 55 </td></tr><tr><td> 1:64 </td><td> 0.053, 0.066 </td><td> 0.059 </td><td> 0.017 </td><td> 12 </td></tr><tr><td> 1:128 </td><td> 0.044, 0.045 </td><td> 0.045 </td><td> 0.003 </td><td> 2 </td></tr></tbody></table><img src="https://www.jove.com/files/ftp_upload/1923/1923fig1.jpg" alt="그림 1" /> 그림 1. 고전적인 보완 경로의 활성화. 고전 경로가 대상 세포의 표면에 결합 면역 글로불린 - M (IgM) 또는 면역 글로불린 - G (IgG)에 의해 활성화됩니다. AB의 FC 부분은 C1q에 바인딩, C1r가 활성화되고 이것은 차례로 함께 C1s 두 분자를 활성화 C1r의 다른 분자를 활성화합니다. C1s 지금도 죽습니다 C2에 대한 구속력이 사이트를 노출 C4를 클리브스. C4b 및 C2A의 바인딩은 C3의 convertase로 불리는 복잡한 형성에 이르게한다. 이 복잡​​한 이제 C5의 convertase을 형성 C3의 convertase와 결합 일부의 C3 성형 C3a와 C3b를 클리브스. 이 복잡​​한 이제 막 공격 복잡한 (MAC)의 형성을 시작 C6에 바인딩 결과 C5b과 C5의 역할을합니다. C8 설정 행위의 궁극적 C9에 대한 최종 MAC의 형성에 결과 C7에 C5b6 행위. (Goldsby 외, 2003). <img src="https://www.jove.com/files/ftp_upload/1923/1923fig2.jpg" alt="그림 2" /> 그림 2. 샘플 데이터하려하고 제어 및 테스트 혈청 샘플에 대한 CH50을 계산. (A), 컨트롤 (•) 및 테스트 () 혈청 샘플을 모두 계산된 비율 (%)가 용해는 희석 요인이 역모를 꾸몄다됩니다. 이 그래프 라인과 교차 다음 수직선이 희석로 그려진까지 (B) 50 % 용해를 계산하려면, 라인은 50 % %의 가치에서 도출됩니다. 이 예제에서는 테스트 혈청의 제어 및 21.6 배 희석의 35 배 희석은 50 %의 용해를 달성해야합니다. 이 데이터는 50 %의 용해에 도달되기 전에 덜 희석 필요한 컨트롤에 비해 테스트 혈청에있는 적은 보완이있다는 것을 나타냅니다. 컨트롤 샘플도 1시 8분 희석시&gt; 100 % 용해를 보여줍니다. 이것은 대부분 보완 구성 요소로 증류수와보다 효율적으로 용해하여 SRBC의 불완전한 용해에 의한 것입니다.

<ol>
	<li>Goldsby, R. A., Kindt, T. J., Osborne, B. A., Kuby, 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> Immunology. , (2003).</li></ol>

CH 50 검정은 많은 interferences의 적용을받습니다. 일부 SRBC는 활동을 보완하기 위해 무관 자연 haemolysis의 결과로, 다른 사람보다 더 연약하고 있습니다. 토끼 항체의 친화도는 많은에서 많은 한 제조 업체에서 다른 다릅니다, 이것은 SRBC에 바인딩 항체의 양을 영향을 미칩니다. 또한, 항체 코팅 SRBC의 다양한 양의와 세포 항체 결과 SRBC을 sensitizing의 과정. 표본 수집 및 저장 오류의 중요한 잠?...

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		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>Sheep red blood cells</td>
<td>&nbsp;</td>
<td>CSL</td>
<td>2490201</td>
<td>Use at 1% final</td>
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<tr>
<td>Serum</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Human serum</td>
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<td>Rabbit Anti-Sheep Haemolytic serum (RBCs), Unconjugated</td>
<td>&nbsp;</td>
<td>AdB Serotec</td>
<td>C12HSB</td>
<td>&nbsp;</td>
<tr>
<td>96 well flat bottom plate</td>
<td>&nbsp;</td>
<td>Sarstedt</td>
<td>83.1839</td>
<td>&nbsp;</td>
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<td>Veronal buffered saline</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Use at 1x final</td>
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<td>Waterbath</td>
<td>&nbsp;</td>
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<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>37&deg;C room/incubator</td>
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measuring the 50% haemolytic complement (ch50) activity of serum

보완 시스템은 세포 용해를 대상으로 이끌 활성화되고 opsonisation 통해 phagocytosis을 용이하게 단백질의 그룹입니다. 개인 보완 구성 요소가 있지만 이것은 경로의 활동에 같은 정보를 제공하지 않습니다 계량하실 수 있습니다. CH<sub&gt; 50</sub&gt; 클래식 보완 경로 (그림 1)의 정품 인증에 대한 심사 분석하고 그것은 경로의 모든 구성 요소의 감소, 부재 및 / 또는 비활성으로 구분합니다. CH<sub&gt; 50</sub&gt; 토끼 안티 양 적혈구 세포 항체 (haemolysin)로 사전 코팅 양 적혈구 (SRBC)을 lyse하는 고전 경로의 혈청 보완 구성 요소의 기능 능력을 테스트합니다. 항체 - 코팅 SRBC은 시험 혈청, 보완의 고전 경로가 활성화되고 haemolysis 결과 incubated 때. 보완 구성 요소가 없을 경우, CH<sub&gt; 50</sub&gt; 레벨 제로합니다, 전통적인 경로 중 하나 이상의 구성 요소가 감소하는 경우, CH<sub&gt; 50</sub&gt;가 감소됩니다. 최적 sensitized SRBC의 고정 볼륨은 각 혈청 희석에 추가됩니다. 부화 후, 혼합물은 centrifuged되고 haemolysis의 정도는 540nm에서 뜨는로 출시 헤모글로빈의 흡광도를 측정하여 계량입니다. 보완 활동의 금액은 항체 코팅 SRBC을 lyse에 혈청 검사의 다양한 dilutions의 용량을 검사에 의해 결정됩니다. 이 비디오는 고전적인 보완 경로의 보완 활동의 수준을 분석에 관련된 실험 단계를 설명합니다.

Watch this Scientific Journal Video about Measuring the 50% Haemolytic Complement CH50 Activity of Serum at JoVE.com

Measuring the 50% Haemolytic Complement CH50 Activity of Serum

고전 경로는 항체에 의해 활성화되고 대상 세포 용해에 culminates 있습니다. CH<sub&gt; 50</sub&gt; 검정은 혈청 샘플의 보완 활동의 측정을 제공합니다. 이 비디오는 CH를 결정에 관련된 단계를 설명합니다<sub&gt; 50</sub&gt; 혈청 샘플의 결과의 계산 및 해석.

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 ...

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JoVE Journal

Immunology and Infection

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

37.6K 조회수.  University of South Australia. 고전 경로는 항체에 의해 활성화되고 대상 세포 용해에 culminates 있습니다. CH 50 검정은 혈청 샘플의 보완 활동의 측정을 제공합니다. 이 비디오는 CH를 결정에 관련된 단계를 설명합니다 50 혈청 샘플의 결과의 계산 및 해석.

비디오: Measuring the 50% Haemolytic Complement CH50 Activity of Serum

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

Determining the Reactivity and Titre of Serum using a Haemagglutination Assay

Haemagglutination is a specific form of agglutination and is used when antibodies bind to red blood cells, which act as a particulate antigen. Red blood cells are particularly useful targets as they are readily available and agglutination is observable using the naked eye. This technique is commonly used to determine the titre of an antibody (Ab), for blood grouping and viral quantification. In this video, the steps involved in preparing and performing a haemagglutination assay is demonstrated using antibodies specific to blood group A-antigens added to red blood cells (Revercells). The antiserum is serially diluted in a 96 well U-bottom microtitre tray, to which is added a suspension of Revercells. The samples are mixed and then incubated at 37&deg;C for 60 minutes. After this time, the samples can then be easily scored for  ve, +ve and intermediate (-/+) haemagglutination reactions. This approach allows for the reactivity and titre of a serum sample to be assessed using a rapid and simple technique. The video will cover the theory behind the assay, how the results are read and interpreted, how the titre is determined, how the assay can be modified and any issues associated with the use of this technique.

Haemagglutination is a form of agglutination where antibodies bind to red blood cells. Red blood cells are both readily available and the results are readily observable using the naked eye. This video demonstrates the steps involved in a haemagglutination assay, interpreting the results and determining the titre.

Haemagglutination 분석을 사용하여 혈청의 반응성 및 Titre를 결정

Haemagglutination is a specific form of agglutination and is used when antibodies bind to red blood cells, which act as a particulate antigen. Red blood ...

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression 1. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis 1. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. 2 In this video, calpain activity in fixed and living murine 32Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32Dkit cells are shown to have elevated activity compared to normal splenocytes.

This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.

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 Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of Listeria, which results in systemic infection2. After injection, Listeria quickly disseminates to the spleen and liver due to uptake by CD8&alpha;+ dendritic cells and Kupffer cells3,4. Once phagocytosed, various bacterial proteins enable Listeria to escape the phagosome, survive within the cytosol, and infect neighboring cells5. During the first three days of infection, different innate immune cells (e.g. monocytes, neutrophils, NK cells, dendritic cells) mediate bactericidal mechanisms that minimize Listeria proliferation. CD8+ T cells are subsequently recruited and responsible for the eventual clearance of Listeria from the host, typically within 10 days of infection6.
Successful clearance of Listeria from infected mice depends on the appropriate onset of host immune responses6	. There is a broad range of sensitivities amongst inbred mouse strains7,8. Generally, mice with increased susceptibility to Listeria infection are less able to control bacterial proliferation, demonstrating increased bacterial load and/or delayed clearance compared to resistant mice. Genetic studies, including linkage analyses and knockout mouse strains, have identified various genes for which sequence variation affects host responses to Listeria infection6,8-14. Determination and comparison of infection kinetics between different mouse strains is therefore an important method for identifying host genetic factors that contribute to immune responses against Listeria. Comparison of host responses to different Listeria strains is also an effective way to identify bacterial virulence factors that may serve as potential targets for antibiotic therapy or vaccine design.
We describe here a straightforward method for measuring bacterial load (colony forming units [CFU] per tissue) and preparing single-cell suspensions of the liver and spleen for FACS analysis of immune responses in Listeria-infected mice. This method is particularly useful for initial characterization of Listeria infection in novel mouse strains, as well as comparison of immune responses between different mouse strains infected with Listeria. We use the Listeria monocytogenes EGD strain15 that, when cultured on blood agar, exhibits a characteristic halo zone around each colony due to &beta;-hemolysis1 (Figure 1). Bacterial load and immune responses can be determined at any time-point after infection by culturing tissue homogenate on blood agar plates and preparing tissue cell suspensions for FACS analysis using the protocols described below. We would note that individuals who are immunocompromised or pregnant should not handle Listeria, and the relevant institutional biosafety committee and animal facility management should be consulted before work commences.

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes is a model organism for studying immune responses and genetic susceptibility to intracellular bacteria in mice. This method enables one to measure bacterial load and generate single-cell suspensions of the liver and spleen from mice for FACS analysis to determine changes in immune cells due to Listeria infection.

감염된 마우스에 세균 하중과 면역 응답 측정 리스테리아 monocytogenes</em

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of ...

Determining the Phagocytic Activity of Clinical Antibody Samples

Antibody-driven phagocytosis is induced via the engagement of Fc receptors on professional phagocytes, and can contribute to both clearance as well as pathology of disease. While the properties of the variable domains of antibodies have long been considered critical to in vivo function, the ability of antibodies to recruit innate immune cells via their Fc domains has become increasingly appreciated as a major factor in their efficacy, both in the setting of recombinant monoclonal antibody therapy, as well as in the course of natural infection or vaccination1-3.
Importantly, despite its nomenclature as a constant domain, the antibody Fc domain does not have constant function, and is strongly modulated by IgG subclass (IgG1-4) and glycosylation at Asparagine 2974-6. Thus, this method to study functional differences of antigen-specific antibodies in clinical samples will facilitate correlation of the phagocytic potential of antibodies to disease state, susceptibility to infection, progression, or clinical outcome.
Furthermore, this effector function is particularly important in light of the documented ability of antibodies to enhance infection by providing pathogens access into host cells via Fc receptor-driven phagocytosis7. Additionally, there is some evidence that phagocytic uptake of immune complexes can impact the Th1/Th2 polarization of the immune response8.
Here, we describe an assay designed to detect differences in antibody-induced phagocytosis, which may be caused by differential IgG subclass, glycan structure at Asn297, as well as the ability to form immune complexes of antigen-specific antibodies in a high-throughput fashion. To this end, 1 &mu;m fluorescent beads are coated with antigen, then incubated with clinical antibody samples, generating fluorescent antigen specific immune complexes. These antibody-opsonized beads are then incubated with a monocytic cell line expressing multiple Fc&gamma;Rs, including both inhibitory and activating. Assay output can include phagocytic activity, cytokine secretion, and patterns of Fc&gamma;Rs usage, and are determined in a standardized manner, making this a highly useful system for parsing differences in this antibody-dependent effector function in both infection and vaccine-mediated protection9.

We present a high-throughput flow cytometric assay to determine the phagocytic activity of antigen-specific antibodies from clinical samples, utilizing fluorescent antigen-coated beads and a monocytic cell line expressing multiple Fc receptors&#x2014;providing receptor usage and phagocytic activity determinations in a standardized and reproducible fashion for any antigen of interest.

임상 항체 샘플 Phagocytic 활동 결정

Antibody-driven phagocytosis is induced via the engagement of Fc receptors on professional phagocytes, and can contribute to both clearance as well as ...

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 ...

Measuring Local Anaphylaxis in Mice

Allergic responses are the result of the activation of mast cells and basophils, and the subsequent release of vasoactive and proinflammatory mediators. Exposure to an allergen in a sensitized individual can result in clinical symptoms that vary from minor erythema to life threatening anaphylaxis. In the laboratory, various animal models have been developed to understand the mechanisms driving allergic responses. Herein, we describe a detailed method for measuring changes in vascular permeability to quantify localized allergic responses. The local anaphylaxis assay was first reported in the 1920s, and has been adapted from the technique published by Kojima et al. in 20071. In this assay, mice sensitized to OVA are challenged in the left ear with vehicle and in the right ear with OVA. This is followed by an intravenous injection of Evans Blue dye. Ten min after injecting Evans Blue, the animal is euthanized and the dye that has extravasated into the ears is extracted overnight in formamide. The absorbance of the extracted dye is then quantified with a spectrophotometer. This method reliably results in a visual and quantifiable manifestation of a local allergic response.

Allergic responses, characterized by the activation of mast cells and basophils, are driven by the cross-linking of IgE and release of proinflammatory mediators. A quantitative assessment of allergic responses can be achieved by using Evans Blue dye to monitor changes in vascular permeability after allergen challenge.

마우스에 현지 아나필락시스 측정

Allergic responses are the result of the activation of mast cells and basophils, and the subsequent release of vasoactive and proinflammatory mediators. ...

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

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

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

인간의 혈액에서 측정 과립구와 단핵구 식균 작용 및 산화 버스트 활동

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

Assays for Studying the Role of Vitronectin in Bacterial Adhesion and Serum Resistance

Bacteria utilize complement regulators as a means of evading the host immune response. Here, we describe protocols for evaluating the role vitronectin acquisition at the bacterial cell surface plays in resistance to the host immune system. Flow cytometry experiments identified human plasma vitronectin as a ligand for the bacterial receptor outer membrane protein H of Haemophilus influenzae type f. An enzyme-linked immunosorbent assay was employed to characterize the protein-protein interactions between purified recombinant protein H and vitronectin, and binding affinity was assessed using bio-layer interferometry. The biological importance of the binding of vitronectin to protein H at the bacterial cell surface in evasion of the host immune response was confirmed using a serum resistance assay with normal and vitronectin-depleted human serum. The importance of vitronectin in bacterial adherence was analyzed using glass slides with and without vitronectin coating, followed by Gram staining. Finally, bacterial adhesion to human alveolar epithelial cell monolayers was investigated. The protocols described here can be easily adapted to the study of any bacterial species of interest.

This report describes protocols for characterizing interactions between bacterial outer membrane proteins and the human complement regulator vitronectin. The protocols can be used to study the binding reactions and biological function of vitronectin in any bacterial species.

세균성 접착 및 혈 청 저항에 Vitronectin의 역할을 공부에 대 한 분석

Bacteria utilize complement regulators as a means of evading the host immune response. Here, we describe protocols for evaluating the role vitronectin ...

50 % Haemolytic 보완 (CH 측정 50) 활동

50 % Haemolytic 보완 (CH 측정_{50) 활동}