우리는 친절 tmuCD40 - 리간드 - 헬라 세포 라인을 제공 클레멘스 박사 Wendtner 감사합니다.

splenocytes에서 murine CD40 - 활성화된 B 세포 (mCD40B)의 생성을 위해 프로토콜은 두 부분으로 나누어져있다 : 부품으로 사용됩니다 판 - 헬라 세포 (tmuCD40L 헬라)를 표현 murine CD40 리간드 (CD40L)의 준비를 보여줍니다 피더 세포를 바운드. 파트 B는 실제 murine CD40 - B 문화를 설명합니다. 피더 세포의 A. 준비 (tmuCD40L 헬라) tmuCD40L 헬라 완전히 합류가해서는 안 자기편 인간 상피 세포주이다. 세포 따라서 주당 두번 splitted 수 있습니다. 6 개 이상 주 동안 Culturing하지 않는 것이 좋습니다. <ol><li> 멸균 피펫으로 기본 문화 올드 미디어를 제거하고 1X PBS의 10 ML과 세포를 씻으십시오. 세척 후 PBS를 대기음. </li><li> 37 10-15분에 75cm 2 플라스크에 1X 트립신 / EDTA (에틸렌 다이아 민 테트라 초산) 4 ML 추가 ° C. 세포를 분리하기 위해 부드러운 도청을 사용합니다. </li><li> 야생 유형 매체 10 ML을 추가하고 트립신과 소화를 중지 부드럽게 회전. </li><li> 멸균 피펫과 50 ML 튜브에 세포 현탁액을 전송 7 분 동안 265 XG에서 세포를 스핀 다운. </li><li> 뜨는을 제거하고 야생 유형 매체 10 ML에있는 세포 펠렛을 resuspend. 세포 현탁액의 나누어지는의 휴대폰 번호를 카운트하고 세포의 적절한 수의 세 50 ML 튜브를 준비 : <ol><li> subculture 2.5 X 10 6 세포 </li><li> murine CD40 - B 세포 배양에 사용 / 잘 조사를위한 0.4 X 10 6 세포 </li><li> 프리즈에 나머지 (필요한 경우). </li></ol></li><li> 7분에 대한 265 XG에서 세포를 스핀 다운. </li><li> 뜨는을 제거합니다. <ol><li> subculturing 들면 : resuspend 2.5 75cm 2 세포 배양 플라스크 10 ML 선택 매체 (세포 밀도 2.5 X 10 5 셀 / ML) 37에서 세포를 품어 X 10 6 세포 ° C와 5 % CO 2. 두 당 주 세포를 분리. </li><li> murine CD40 - B 세포 배양의 경우 : 한 6 자 접시 2.4 X 10 6 세포가 필요합니다. 0.2 X 10 6 세포 / ML의 밀도에 야생 유형의 매체에 tmuCD40L 헬라 세포를 Resuspend하고 78 쥐에서 그들을 비추다. 물론 각 37에서 그들을 품어 ° C와 5 % CO 2에 세포 현탁액의 플레이트 2 ML. tmuCD40L 헬라 세포가 자기편 때 B - 세포 자극이 준비한 접시를 사용합니다 (최소한 4 시간 : 현미경을 사용하여 준수를 확인, B - 세포 자극을 시작하는 이상 24 H를 기다리지 않는다). (B. 계속) </li></ol></li></ol> B. Murine CD40 - B 세포 배양 CD40 - 자극 (일 공)에 대한 splenocytes의 I. 준비 : 주의 : 당신이 피더 세포가 자기편 것을 확인할 계속하기 전에. 항상 murine 인터루킨 - 4, β - 메르 캅 토 에탄올과 성장 매체 시클 로스 포린 바로 사용하기 전에 새로운 솔루션을 추가합니다. <ol><li> C57BL / 6 마​​우스 (haplotype H - 2B)의 비장을 절개하다하고 10-20 ML DMEM에 두번 씻어 다른 한 튜브에서 10mL 피펫 팁로 전송하여 Gentamycin [15μg/mL]로 보충. 마우스의 전송 오염 물질 (예 : 머리카락)하지 마십시오. 셀 스트레이너 (100μm)를 통해 전달하여 비장을 말하다 및 매체의 또 다른 10 ML와 스트레이너를 헹굴. 상온에서 7 분 265 XG에 splenocytes를 스핀 다운, 7mL muCD40 - B의 세포 매체 및 Ficoll 밀도 원심 분리하여 별도의 lymphocytes들을 resuspend. 그렇게 30 분 450 XG에 15 ML 튜브와 원심 분리기 5 ML 마우스 Pancoll (실온에서 preheated)에 레이어 셀을 상온에서 브레이크없이 할 수 있습니다. </li><li> 인터페이스에서 그대로 림프구 계층을 떠나, 상위 레이어의 대부분을 그립니다. 새로운 50 ML 튜브에 림프구 계층을 전송, 기타 세포를 제거 7 분 265 XG에 가서 회전에 의해 Gentamycin [15μg/mL]와 30mL DMEM과 함께 한 번 씻는다. 뜨는을 취소하고 mCD40 - B 세척 매체 10 ML에있는 세포를 resuspend. 세포 현탁액의 나누어지는의 휴대폰 번호를 확인합니다. 세포 현탁액의 나누어지는를 세어 휴대폰 번호를 확인합니다. </li><li> 7분에 대한 265 XG에서 세포의 필요한 양을 스핀 다운. 6 잘 플레이트 5 X 10 6 세포 / 잘 따라서 판 30 X 10 6 셀 필요합니다. </li><li> 뜨는를 제거하고 성장 요소로, 시클 로스 포린 100 μm의 β - 메르 캅 토 에탄올 및 0.63 μg / ML은 방지하기 위해 갓 1 U / 인터루킨 - 4의 ML과 보충 mCD40 - B 문화 매체 1.25 X 10 6 세포 / ML에서 lymphocytes을 resuspend T - 세포의 가지 (감안할 때 농도가 한 ML 문화 매체를 참조!). </li><li> 6 잘 플레이트 tmuCD40L 헬라 세포로 사전 incubated에서 뜨는을 제거합니다. </li><li> 부드럽게 6 잘 접시의 각각 잘 수있는 림프구 정지 4 ML (1.25 X 10 6 세포 / ML)를 추가합니다. </li><li> 37 접시를 품어 ° C 5 % CO 2. </li&gt; <li> 일 3 전지 (II.1 계속.) reculture. </li></ol> II. mCD40B 세포 (3 일 후 일주일에 두 번)의 Subculture : <ol><li> 노골적인 협상을 50 ML 튜브에 10 ML의 피펫과 수영장 그들과 함께 위아래로 pipetting하여 수확 mCD40B 전지 클러스터. </li><li> 7분에 대한 265 XG에 스핀 다운 및 완전히 mCD40 - B 세척 매체로 뜨는 교체하십시오. 나누어지는의 세포 금액을 계산하는 동안, 7 분 동안 265 XG에서 세포를 스핀 다운. 0.75 X 10 6 세포 / ML의 농도에서 mCD40 - B 문화 매체 Resuspend mCD40B 세포. </li><li> 1 U / ML, 100 μm의 β - 메르 캅 토 에탄올의와 매체 시클 로스 포린 0.63 μg / ML의 농도에서 인터루킨 - 4 murine의 새로운 솔루션을 추가합니다. </li><li> 6 잘 플레이트 조사 tmuCD40L 헬라 세포로 사전 incubated에서 뜨는을 제거합니다. </li><li> 부드럽게 6 잘 접시의 각각 잘 수있는 mCD40B 세포 현탁액 (0.75 X 10 6 세포 / ML) 4 ML를 추가합니다. </li><li> 37 접시를 품어 ° C 5 % CO 2. </li><li> 다시 모든 3~4일 14 일 총 후에 매우 순수 murine CD40 - B 세포 활성화와 최종 수있는 세포가 subculture. </li></ol> C.의 문제 해결 : 어떤 경우 CD40 - B 세포 성장하지? <ol><li> 당신은 피더 세포의 CD40 리간드 발현에 대한 검사 있습니까? </li><li> 24 세 이상 자극에 사용되는 플레이트 - 바운드 피더 세포 있습니까? </li><li> mycoplasma와 오염이 있나요? </li><li> 보완을 위해 사용되는 인터루킨 - 4 솔루션은 신선한 해동이고 이것은 적절한 생물 학적 활동을 있었나요? </li><li> β - 메르 캅 토 에탄올과 시클 로스 포린 A는 정확한 농도에 추가습니까? </li></ol><img src="/files/ftp_upload/1734/1734fig1a.jpg" alt="그림 1" /> 그림 1A. <img src="/files/ftp_upload/1734/1734fig1b.jpg" alt="그림 1 B" /> 그림 1B. <img src="/files/ftp_upload/1734/1734fig1d.jpg" alt="그림 1 D" /> 그림 1D. <img src="/files/ftp_upload/1734/1734fig2.jpg" alt="그림 2" /> 그림 2.

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실험은 실험실 동물 관리 방법에 대한 국내 및 유럽 지침에 따라 수행되었다. 독일의 동물 복지 법률을 참조하면 동물이 적절한 당국에 의해 정기적으로 제어되었다.

이 프로토콜의 수정은 특히도 murine CD40가 활성화된 B 세포의 효율적인 세대에 항복, CD40 - 자극 및 사용 마우스 변형의 유형의 기원에 대해 가능합니다. 아마디 외. CD40 - 리간드와 transfected murine 마우스 섬유아 세포에 대한 유사한 결과를 보여주었다. 외부의 항원이 맞는 프레 젠 테이션에서 확실하게 제외하지만, 보안 및 생체내의 독성에 관한 집중 연구는 인간의 transfected 헬라 세포를 사용?...

미디어 A. 준비 : <table border="1"><tbody><tr><td> 피더 세포 야생 유형 매체 </td><td> 피더 셀 선택 매체 </td><td></td></tr><tr><td> 시약 </td><td> 집중 </td><td> 시약 </td><td> 집중 </td></tr><tr><td> RPMI 1640 </td><td></td><td> RPMI 1640 </td><td></td></tr><tr><td> L - 글루타민 </td><td> 300 μg / ML </td><td> L - 글루타민 </td><td> 300 μg / ML </td></tr><tr><td> FBS </td><td> 10% </td><td> FBS </td><td> 10% </td></tr><tr><td> HEPES </td><td> 10 MM </td><td> HEPES </td><td> 10 MM </td></tr><tr><td> Gentamycin </td><td> 15 μg / ML </td><td> Gentamycin </td><td> 15 μg / ML </td></tr><tr><td></td><td></td><td> Hygromycin B </td><td> 0.2 MG / ML </td></tr></table><table border="1"><tbody><tr><td> mCD40 - B 세척 매체 </td><td> mCD40 - B 문화 매체 </td></tr><tr><td> 시약 </td><td> 집중 </td><td> 시약 </td><td> 집중 </td></tr><tr></tr><tr><td> D - 멤 </td><td> </td><td> D - 멤 </td><td> </td></tr><tr><td> L - 글루타민 </td><td> 580 μg / ML </td><td> L - 글루타민 </td><td> 580 μg / ML </td></tr><tr><td> 포도당 </td><td> 4.5 MG / ML </td><td> 포도당 </td><td> 4.5 MG / ML </td></tr><tr><td> HEPES </td><td> 10 MM </td><td> HEPES </td><td> 10 MM </td></tr><tr><td> Gentamycin </td><td> 15 μg / ML </td><td> Gentamycin </td><td> 15 μg / ML </td></tr><tr><td> </td><td> </td><td> 멤 </td><td> 0.1mM (1X) </td></tr><tr><td> </td><td> </td><td> FBS </td><td> 10% </td></tr></tbody></table> B. 기타 시약 : <table border="1"><tr><td> 시약 </td><td> 회사 </td><td> 아니오를 주문하지 </td></tr><tr><td> RPMI 1640 </td><td> Invitrogen Gibco </td><td> REF 21,875 </td></tr><tr><td> D - 멤 </td><td> Invitrogen Gibco </td><td> REF 41,965 </td></tr><tr><td> Dulbeccos PBS (10X) </td><td> PAA </td><td> 고양이도없는 H15 - 011 </td></tr><tr><td> Gencin </td><td> 델타 선택 </td><td> 예술 없음 7,395,800 </td></tr><tr><td> FBS </td><td> LONZA </td><td> 고양이도없는 DE14 - 802C </td></tr><tr><td> HEPES 버퍼 </td><td> PAA </td><td> 고양이도없는 S11 - 001 </td></tr><tr><td> 멤 NEAA </td><td> Invitrogen Gibco </td><td> REF 11,140 </td></tr><tr><td> Hygromycin B </td><td> PAA </td><td> 고양이도없는 P02 - 015 </td></tr><tr><td> 인터루킨 - 4 재조합 Murine </td><td> Immunotools </td><td> 고양이도없는 12,340,045 </td></tr><tr><td> 시클 로스 포린 </td><td> 노바티스 </td><td> 고양이도없는 NDC 0078-0109-01 </td></tr><tr><td> 트립신 / EDTA (에틸렌 다이아 민 테트라 초산) (10X) </td><td> Invitrogen Gibco </td><td> REF 15400-054 </td></tr></table> C. 기타 용품 : <table border="1"><tbody><tr><td> 시약 </td><td> 회사 </td><td> 아니오를 주문하지 </td></tr><tr><td> 멸균 피펫 팁 </td><td> Sarstedt </td><td></td></tr><tr><td> 6 - 웰 플레이트 </td><td> NUNC </td><td> 고양이도 140,675 </td></tr><tr><td> 50mL 원뿔 관 </td><td> BD 팰컨 ™ </td><td> 고양이도 352,070 </td></tr><tr><td> 조직 문화 플라스크 </td><td> Sarstedt </td><td> 고양이도없는 83.1813.002 </td></tr></tbody></table>

murine model of cd40-activation of b cells

B 세포에 관한 연구는 CD40 활성화들은 항원 프레 젠 테이션 능력을 향상하는 것으로 나타났습니다. 인터루킨 - 4 및 CD40 리간드 (CD40L)와 자극을 때, 인간의 B 세포는 고도로 순수 CD40 - B 세포 (매우 큰 금액을 14 일 이내에 말초혈 소량의 어려움없이 확장 가능&gt; 10<sup&gt; 9</sup건강한 기증자뿐만 아니라 암 환자에서 환자마다&gt; 전지)<sup&gt; 1-4</sup&gt;. CD40 - B 세포 중요한 림프절 유도 분자를 표현하고 T 세포를 유도할 수<em&gt; 체외에서</em<sup&gt; 5</sup&gt;. 또한 그들은 효율적으로 T 세포, 프로세스와 현재의 항원을 차지<sup&gt; 6,7</sup&gt;. CD40 - B 세포는 주요 순진하지에만 표시뿐만 아니라 메모리 T 세포를 확장했다<sup&gt; 8,9</sup&gt;. 따라서 CD40 - 활성 B 세포 (CD40 - B 세포)의 대체 소스로 연구되었습니다 단백질 stimulatory 항원 제시 세포 기반 immunotherapy1, 5,10를위한 세포 (APC)를. CD40 - B 세포가 생체내 효과적인 T 세포 반응을 유도하고 우리가 murine CD40 - 활성 B 세포의 생성을위한 세포 배양 시스템을 확립 기본 메커니즘을 연구 여부를 진학하기 위해서는. splenocytes를 사용하거나 CD40 - 정품 인증 C57BL / 6 마​​우스에서 B 세포를 정화, 최적의 조건은 다음과 같이 확인되었습니다 : C57BL / 6 마​​우스 (haplotype H - 2B) lymphocytes의 splenocytes의 시작은 밀도 기울기 원심 분리와 헬라와 함께 공동 양식에 의해 정화 아르 재조합 murine CD40 리간드 (tmuCD40L 헬라)를 표현 세포<sup&gt; 11</sup&gt;. 세포 3-4일 모든 recultured하고 같은 CD40L, 인터루킨 4와 같은 주요 구성 요소는 - 메르 캅 토 에탄올과 시클 로스 포린 A은 보충함 수 있습니다. 이 프로토콜에서는 인간 CD40 - B 세포 (그림 1A, B)에 APC - 표현형 유사한 완벽하게 활성화 murine CD40 - B 세포 (mCD40B)을 구하는 방법을 보여줍니다. CD40 - 자극은 세포 배양 후 14 일 이내에 신속한 가지 고도 순수 (&gt; 90%) CD19 + B 세포의 확장 (그림 1C, D)에 연결됩니다. 비 transfected 세포와 오염을 방지하려면 transfectants에 murine CD40 리간드 표현 (그림 2) 정기적으로 제어되어야한다. Murine CD40 - 활성 B 세포는 체외에서 생체내의 APC로 함수 자신의 잠재력을 조사하기 위해 B - 세포 활성화와 차별화뿐만 아니라 공부를하는 데 사용할 수 있습니다. 또한, 그들은 종양에 대한 치료 또는 예방 접종을 수립을위한 유망한 도구를 대표하고 안전과이 방법의 immunogenicity에 관한 질문에 대답하는 데 도움이됩니다<sup&gt; 12</sup&gt;.

Watch this Scientific Journal Video about Murine Model of CD40-activation of B cells at JoVE.com

Murine Model of CD40-activation of B cells

이 비디오에서는, 우리는 CD40 - 활성화 및 면역의 유도를 연구하는 모델 항원 제시 세포 (APC)로 사용할 수 있습니다 C57BL / 6 마​​우스 splenocytes에서 murine B 세포의 확장 프로 시저를 보여줍니다.

B 세포의 CD40 - 정품 인증 Murine 모델

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

murine-model-of-cd40-activation-of-b-cells

Research

JoVE Journal

Immunology and Infection

12.5K 조회수.  University Hospital of Cologne. 이 비디오에서는, 우리는 CD40 - 활성화 및 면역의 유도를 연구하는 모델 항원 제시 세포 (APC)로 사용할 수 있습니다 C57BL / 6 마​​우스 splenocytes에서 murine B 세포의 확장 프로 시저를 보여줍니다.

비디오: Murine Model of CD40-activation of B cells

Induction and Assessment of Class Switch Recombination in Purified Murine B Cells

Humoral immunity is the branch of the immune system maintained by B cells and mediated through the secretion of antibodies. Upon B cell activation, the immunoglobulin locus undergoes a series of genetic modifications to alter the binding capacity and effector function of secreted antibodies. This process is highlighted by a genomic recombination event known as class switch recombination (CSR) in which the default IgM antibody isotype is substituted for one of IgG, IgA, or IgE. Each isotype possesses distinct effector functions thereby making CSR crucial to the maintenance of immunity. Diversification of the immunoglobulin locus is mediated by the enzyme activation-induced cytidine deaminase (AID). A schematic video describing this process in detail is available online (http://video.med.utoronto.ca/videoprojects/immunology/aam.html). AID's activity and the CSR pathway are commonly studied in the assessment of B cell function and humoral immunity in mice. The protocol outlined in this report presents a method of B cell isolation from murine spleens and subsequent stimulation with bacterial lipopolysaccharide (LPS) to induce class switching to IgG3 (for other antibody isotypes see Table 1). In addition, the fluorescent cell staining dye Carboxyfluorescein succinimidyl ester (CFSE) is used to monitor cell division of stimulated cells, a process crucial to isotype switching 1, 2. The regulation of AID and the mechanism by which CSR occurs are still unclear and thus in vitro class switch assays provide a reliable method for testing these processes in various mouse models. These assays have been previously used in the context of gene deficiency using knockout mice 3. Furthermore, in vitro switching of B cells can be preceded by viral transduction to modulate gene expression by RNA knockdown or transgene expression 4-6. The data from these types of experiments have impacted our understanding of AID activity, resolution of the CSR reaction, and antibody-mediated immunity in the mouse.

Following antigen exposure, subpopulations of activated B cells undergo a process known as class switch recombination (CSR) to produce antibody isotypes with distinct effector functions. The protocol outlined in this report explains how CSR can be induced and analyzed in vitro for the purposes of studying B cell function.

Murine B 세포를 정화에 유도 및 클래스 스위치 재조합 평가

Humoral immunity is the branch of the immune system maintained by B cells and mediated through the secretion of antibodies. Upon B cell activation, the ...

연구

면역학 및 감염병학

Recombinant Retroviral Production and Infection of B Cells

The transgenic expression of genes in eukaryotic cells is a powerful reverse genetic approach in which a gene of interest is expressed under the control of a heterologous expression system to facilitate the analysis of the resulting phenotype. This approach can be used to express a gene that is not normally found in the organism, to express a mutant form of a gene product, or to over-express a dominant-negative form of the gene product. It is particularly useful in the study of the hematopoetic system, where transcriptional regulation is a major control mechanism in the development and differentiation of B cells 1, reviewed in 2-4.
Mouse genetics is a powerful tool for the study of human genes and diseases. A comparative analysis of the mouse and human genome reveals conservation of synteny in over 90% of the genome 5. Also, much of the technology used in mouse models is applicable to the study of human genes, for example, gene disruptions and allelic replacement 6. However, the creation of a transgenic mouse requires a great deal of resources of both a financial and technical nature. Several projects have begun to compile libraries of knock out mouse strains (KOMP, EUCOMM, NorCOMM) or mutagenesis induced strains (RIKEN), which require large-scale efforts and collaboration 7. Therefore, it is desirable to first study the phenotype of a desired gene in a cell culture model of primary cells before progressing to a mouse model. 
Retroviral DNA integrates into the host DNA, preferably within or near transcription units or CpG islands, resulting in stable and heritable expression of the packaged gene of interest while avoiding transcriptional silencing 8 9. The genes are then transcribed under the control of a high efficiency retroviral promoter, resulting in a high efficiency of transcription and protein production. Therefore, retroviral expression can be used with cells that are difficult to transfect, provided the cells are in an active state during mitosis. Because the structural genes of the virus are contained within the packaging cell line, the expression vectors used to clone the gene of interest contain no structural genes of the virus, which both eliminates the possibility of viral revertants and increases the safety of working with viral supernatants as no infectious virions are produced 10. 
Here we present a protocol for recombinant retroviral production and subsequent infection of splenic B cells. After isolation, the cultured splenic cells are stimulated with Th derived lymphokines and anti-CD40, which induces a burst of B cell proliferation and differentiation 11. This protocol is ideal for the study of events occurring late in B cell development and differentiation, as B cells are isolated from the spleen following initial hematopoetic events but prior to antigenic stimulation to induce plasmacytic differentiation.

An efficient system of structure and function analysis of a gene in an ex vivo culture of splenic B-lymphocytes is described. This method takes advantage of recombinant retroviral production in a helper free, ecotrophic packaging cell line. Stable, heritable expression of a gene of interest within primary lymphocytes is achieved leading to generation of surface antibodies on B cells undergoing class switch recombination.

B 세포의 재조합 생산 및 Retroviral 감염

The transgenic expression of genes in eukaryotic cells is a powerful reverse genetic approach in which a gene of interest is expressed under the control ...

Murine Model of Allergen Induced Asthma

Asthma is a major cause of morbidity and mortality, affecting some 300 million people throughout the world.1 More than 8% of the US population has asthma, with the prevalence increasing.2 As with other diseases, animal models of allergic airway disease greatly facilitate understanding of the underlying pathophysiology, help identify potential therapeutic targets, and allow preclinical testing of possible new therapies. Models of allergic airway disease have been developed in several animal species, but murine models are particularly attractive due to the low cost, ready availability, and well-characterized immune systems of these animals.3 Availability of a variety of transgenic strains further increases the attractiveness of these models.4 Here we describe two murine models of allergic airway disease, both employing ovalbumin as the antigen. Following initial sensitization by intraperitoneal injection, one model delivers the antigen challenge by nebulization, the other by intratracheal delivery. These two models offer complementary advantages, with each mimicking the major features of human asthma.5
The major features of acute asthma include an exaggerated airway response to stimuli such as methacholine (airway hyperresponsiveness; AHR) and eosinophil-rich airway inflammation. These are also prominent effects of allergen challenge in our murine models,5,6 and we describe techniques for measuring them and thus evaluating the effects of experimental manipulation. Specifically, we describe both invasive7 and non-invasive8 techniques for measuring airway hyperresponsiveness as well as methods for assessing infiltration of inflammatory cells into the airways and the lung. Airway inflammatory cells are collected by bronchoalveolar lavage while lung histopathology is used to assess markers of inflammation throughout the organ. These techniques provide powerful tools for studying asthma in ways that would not be possible in humans.

Experimental mouse models of allergic asthma offer new possibilities for studying disease pathogenesis and developing new therapeutics. These models are well suited to measuring factors governing the allergic immune response, airway inflammation, and pulmonary pathophysiology.

Asthma is a major cause of morbidity and mortality, affecting some 300 million people throughout the world.1 More than 8% of the US population has asthma, ...

Detection of True IgE-expressing Mouse B Lineage Cells

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH isotypes, such as IgA, IgE and IgG. Measurement of IgH CSR in vitro is a key method for the study of a number of biologic processes ranging from DNA recombination and repair to aspects of molecular and cellular immunology. In vitro CSR assay involves the flow cytometric measurement surface Ig expression on activated B cells. While measurement of IgA and IgG subclasses is straightforward, measurement of IgE by this method is problematic due to soluble IgE binding to Fc&#949;RII/CD23 expressed on the surface of activated B cells. Here we describe a unique procedure for accurate measurement of IgE-producing mouse B cells that have undergone CSR in culture. The method is based on trypsin-mediated cleavage of IgE-CD23 complexes on cell surfaces, allowing for detection of IgE-producing B lineage cells by cytoplasmic staining. This procedure offers a convenient solution for flow cytometric analysis of CSR to IgE.

In vitro analysis of class switch recombination in mice is challenging due to cytophilic IgE molecules bound to Fc receptors on the surface of B cells. We describe a method for IgE detection using trypsin-mediated cleavage of surface-bound IgE prior to fixation and permeabilization for cytoplasmic fluorescence staining.

진정한 검출 마우스 B 리니지 세포를 IgE 항체가 발현

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH ...

Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, including the development of immunotherapeutics. However, the techniques to identify and produce such antibodies tend to be arduous and sometimes the heavy and light chain pair of the antibodies are dissociated. Here, we describe a relatively simple, straightforward protocol to produce human recombinant monoclonal antibodies from human peripheral blood mononuclear cells using immortalization with Epstein-Barr Virus (EBV) and Toll-like receptor 9 activation. With an adequate staining, B cells producing antibodies can be isolated for subsequent immortalization and clonal expansion. The antibody transcripts produced by the immortalized B cell clones can be amplified by PCR, sequenced as corresponding heavy and light chain pairs and cloned into immunoglobulin expression vectors. The antibodies obtained with this technique can be powerful tools to study relevant human immune responses, including autoimmunity, and create the basis for new therapeutics.

Synthesis of human monoclonal antibodies is the first step in studies aimed at unraveling the pathophysiological mechanisms of auto-antibody-mediated immune responses. We have developed a protocol to generate recombinant human immunoglobulin G (IgG) monoclonal antibodies from blood sorted B cells, including B-cell isolation, antibody cloning and in vitro synthesis.

정렬 된 불멸화 된 B 세포로부터 재조합 인간 IgG 단일 클론 항체의 생성

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, ...

A Murine Model of Group B Streptococcus Vaginal Colonization

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of 10 - 30% of adults. In immune-compromised individuals, including neonates, pregnant women, and the elderly, GBS may switch to an invasive pathogen causing sepsis, arthritis, pneumonia, and meningitis. Because GBS is a leading bacterial pathogen of neonates, current prophylaxis is comprised of late gestation screening for GBS vaginal colonization and subsequent peripartum antibiotic treatment of GBS-positive mothers. Heavy GBS vaginal burden is a risk factor for both neonatal disease and colonization. Unfortunately, little is known about the host and bacterial factors that promote or permit GBS vaginal colonization. This protocol describes a technique for establishing persistent GBS vaginal colonization using a single &#946;-estradiol pre-treatment and daily sampling to determine bacterial load. It further details methods to administer additional therapies or reagents of interest and to collect vaginal lavage fluid and reproductive tract tissues. This mouse model will further the understanding of the GBS-host interaction within the vaginal environment, which will lead to potential therapeutic targets to control maternal vaginal colonization during pregnancy and to prevent transmission to the vulnerable newborn. It will also be of interest to increase our understanding of general bacterial-host interactions in the female vaginal tract.

A Murine Model of Group B Streptococcus Vaginal Colonization

The purpose of this protocol is to imitate human group B Streptococcus (GBS) vaginal colonization in a murine model. This method may be used to investigate host immune responses and bacterial factors contributing to GBS vaginal persistence, as well as to test therapeutic strategies.

그룹 B의 쥐 모델<em&gt; 연쇄상 구균</em&gt; 질 식민

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...

Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate and enrich these rare cells for studies of their phenotype and biology. Approaches are inevitably based on the principle that B cells recognize native antigen by virtue of cell surface receptors that are representative in specificity of antibodies that will eventually be secreted by their differentiated daughters. Perhaps the most obvious approach to the problem involves use of fluorochrome-conjugated antigens in conjunction with fluorescence-activated cell sorting (FACS). However, the utility of these methods is limited by cell frequency and the achievable rate of analysis and isolation by electronic sorting. A novel method to enrich rare antigen-specific B cells using magnetic nanoparticles that results in high yield enrichment of antigen-reactive B cells from large starting cell populations is described. This method enables improved monitoring of the phenotype and biology of antigen reactive cells before and following in vivo antigen encounter, such as after immunization or during development of autoimmunity.

A simple yet effective method that employs magnetic nanoparticles to detect and enrich antigen-reactive B cells for functional and phenotypic analysis is described.

표현형 및 기능 분석에 대한 검색 및 희귀의 농축 항원 특이 적 B 세포

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate ...

Analysis of Shear Flow-induced Migration of Murine Marginal Zone B Cells In Vitro

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, MZBs must migrate up the shear force of blood flow. We present here a method for analyzing this flow-induced MZB migration in vitro. First, MZBs are isolated from the mouse spleen. Second, MZBs are settled on integrin ligands in flow chamber slides, exposed to shear flow, and imaged under a microscope while migrating. Third, images of the migrating MZBs are processed using the MTrack2 automatic cell tracking plugin for ImageJ, and the resulting cell tracks are quantified using the Ibidi chemotaxis tool. The migration data reveal how fast the cells move, how often they change direction, whether the shear flow vector affects their migration direction, and which integrin ligands are involved. Although we use MZBs, the method can easily be adapted for analyzing migration of any leukocyte that responds to the force of shear flow.

Marginal zone B cells (MZBs) respond to the force of shear flow by re-orienting their migration path up the flow. This protocol shows how to record and analyze the migration using a fluidics unit, pump, microscope imaging system, and free software.

분석 Murine 가장자리 지역 B 세포 생체 외에서의 전단 흐름 유도 마이그레이션

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, ...

In Vitro Differentiation Model of Human Normal Memory B Cells to Long-lived Plasma Cells

Plasma cells (PCs) secrete large amounts of antibodies and develop from B cells that have been activated. PCs are rare cells located in the bone marrow or mucosa and ensure humoral immunity. Due to their low frequency and location, the study of PCs is difficult in human. We reported a B to PC in vitro differentiation model using selected combinations of cytokines and activation molecules that allow to reproduce the sequential cell differentiation occurring in vivo. In this in vitro model, memory B cells (MBCs) will differentiate into pre-plasmablasts (prePBs), plasmablasts (PBs), early PCs and finally, into long-lived PCs, with a phenotype close to their counterparts in healthy individuals.&#160;We also built an open access bioinformatics tools to analyze the most prominent information from GEP data related to PC differentiation. These resources can be used to study human B to PC differentiation and in the current study, we investigated the gene expression regulation of epigenetic factors during human B to PC differentiation.

Using multi-step culture systems, we report an in vitro B cell to plasma cell differentiation model.

수명이 긴 플라즈마 세포에 인간의 정상적인 메모리 B 세포의 체 외 분화 모델

Plasma cells (PCs) secrete large amounts of antibodies and develop from B cells that have been activated. PCs are rare cells located in the bone marrow or ...

Isolation of Lamina Propria Mononuclear Cells from Murine Colon Using Collagenase E

The intestine is the home to the largest number of immune cells in the body. The small and large intestinal immune systems police exposure to exogenous antigens and modulate responses to potent microbially derived immune stimuli. For this reason, the intestine is a major target site of immune dysregulation and inflammation in many diseases including but, not limited to inflammatory bowel diseases such as Crohn&#8217;s disease and ulcerative colitis, graft-versus-host disease (GVHD) after bone marrow transplantation (BMT), and many allergic and infectious conditions. Murine models of gastrointestinal inflammation and colitis are heavily used to study GI complications and to pre-clinically optimize strategies for prevention and treatment. Data gleaned from these models via isolation and phenotypic analysis of immune cells from the intestine is critical to further immune understanding that can be applied to ameliorate gastrointestinal and systemic inflammatory disorders. This report describes a highly effective protocol for the isolation of mononuclear cells (MNC) from the colon using a mixed silica-based density gradient interface. This method reproducibly isolates a significant number of viable leukocytes while minimizing contaminating debris, allowing subsequent immune phenotyping by flow cytometry or other methods.

The goal of this protocol is to isolate mononuclear cells that reside in the lamina propria of the colon by enzymatic digestion of the tissue using collagenase. This protocol allows for the efficient isolation of mononuclear cells resulting in a single cell suspension which in turn can be used for robust immunophenotyping.

콜라게나아제 E를 사용하여 뮤린 결장에서 라미나 프로프리아 단핵 세포의 분리

The intestine is the home to the largest number of immune cells in the body. The small and large intestinal immune systems police exposure to exogenous ...