Name: detection of true ige-expressing mouse b lineage cells
Uploaded: 2014-12-01T14:00:00.000+00:00
Duration: 9 min 40 s
Description: Watch this Scientific Journal Video about Detection of True IgE-expressing Mouse B Lineage Cells at JoVE.com

DRW는 NIH 보조금 AI089972과 AI113217,에 의해 점막 면역학 연구 팀에 의해 지원하고, 버로우즈 웰컴 기금 의료 과학자에 대한 경력 상을 보유하고있다.

참고 : 여기에 설명 된 모든 실험은 기관 동물 관리 및 사용위원회 (IACUC) 가이드 라인에 따라했고, 동물 연구 아동 병원 (ARCH), 보스턴, 질량에 의해 승인했다. 시약 1. 준비 <ol><li> 1000 배 스톡 IL-4 : H 2 O 0.1 % 소 혈청 알부민 (BSA)의 20 μg의 / ㎖ 묽은 IL-4 사이토 카인. -20 ℃에서 1.5 ml의 마이크로 원심 분리 튜브 및 상점을 200㎕ 분량 씩 분주한다. </li><li> 1,000 배 안티 CD40 : 4 ℃에서 1.0 ㎎ / ㎖, 가게에서 제조 업체에서 얻습니다. </li><li> B 세포 자극 미디어 : RPMI-1640 배지 425 mL의 추가, 75 갓 해동 소 태아 혈청의 ㎖ (FCS), 1.0 M HEPES 버퍼, 5 ML의 L 글루타민 재고 10 ㎖, 5 ml의 페니실린 : 스트렙토 마이신 주식, 5 2- 머 캅토 에탄올 ml의 최소 필수 중간 비 필수 아미노산 (MEM-NEAA), 3.5 μL. 일주일에 최대 4 ℃에서 둡니다. IL-4 (20 NG / ㎖) 및 항 CD40 (1.0 ㎍ / ㎖)에 사용 직전에 추가단지 필요한 미디어의 볼륨. </li><li> 2 % FCS-FACS 버퍼 : 1X PBS의 490 ml의 FCS의 10 ML을 추가합니다. 4 ℃에서 보관하십시오. </li><li> 배 트립신 EDTA 용액 : 1X PBS 20ml에 10 배 트립신 EDTA 원액 (5.0 g 트립신, 리터 당 2.0 g의 EDTA) 5 mL를 희석. 저장 트립신 EDTA는 2 배에서 4 ° C에서 분주 작업. 사용하기 전에 실온으로 가온시킨다. </li><li> 태아 혈청은 : 최대 2 주 동안 4 ℃에서 해동 FCS를 유지합니다. 세포질 염색 절차 전에 얼음 15 분에 놓습니다. </li><li> 중성 완충 포르말린 : 10 % 포르말린 용액 (3.7 % 파라 포름 알데히드)를 준비한다. 포르말린은 독성이 적절한 PPE를 착용하는 동안 따라서 실험실 흄 후드를 처리 할 수​​ 있습니다. 화학 스토리지 캐비닛을 실온에서 보관 포르말린. </li><li> 메탄올 : -20 ° C에서 보관 절대 메탄올 (100 %)의 세포 투과 가능하게 할 준비가 될 때까지. 그 메탄올이 가연성 적절한 냉동실에 보관해야합니다. </li></ol> 2.정제 된 B 세포의 활성화 <ol><li> 갓 수확 된 마우스의 비장에서 부드럽게 3 ㎖ 주사기의 플런저와 70 μm의 셀 스트레이너를 통해 장기를 눌러 단일 세포 현탁액을 제조. 15 ml의 원추형 원심 분리 관에서, 4 ℃에서 5 분 동안 300 XG에서 1X PBS 10ml에 세포를 수집하고 원심 분리기. </li><li> 5 분 동안 적혈구 용혈 완충액 1 ㎖에 재현 탁 상등액과 펠렛을 따르다. 5 분 동안 300 XG에서 1X PBS 13.5 ml의 원심으로 중화. </li><li> (선택 사항) 자기 (磁 气) 제조업체의 지침에 따라 MACS 정화 프로토콜 다음, 구슬을 방지 CD45R (B220)를 사용하여 B 세포를 표지 된 구분합니다. </li><li> 37 ° C에서 IL-4 (20 NG / ml) 및 오일에 대한 항 CD40 (1.0 ㎍ / ㎖) 함유 가온 B 세포 자극 배지에서 1.0 × 106 세포 / ml으로 정제 된 B 세포를 자극한다. 6 웰 배양 판 (4 ml의 각)의 두 우물로 나누어 문화의 8 ml로 시작합니다. </li><li> cultu 확인1.0 × 106 세포 / ml로 유지하기 위해 매일 재. 다른 웰로 분할 신선한 IL-4와 항 CD40을 함유하는 B 세포 자극 매체 배양을 희석하여 농도를 조정한다. </li></ol> 3. 트립신, 고정 및 투과성으로 <ol><li> 트립신 <ol><li> 5 분 300 XG에, 15 ML 원뿔 튜브에 원심 분리기를 배양 세포의 최대 1.0 × 10 (7)를 수집, 상층 액을 가만히 따르다. </li><li> 셀 수집 미디어에서 잔류 단백질을 제거하기 위해 PBS의 10 ml로 세척 할 것. 원심 분리 단계를 반복하여 상층 액을 가만히 따르다. </li><li> (약 100 μL)을 경사 분리 한 후 잔류 체적의 세포 펠렛을 재현 탁. </li><li> 조심스럽게 세포에 실온 0.1 % (2 배) 트립신 EDTA의 800 μl를 추가합니다. 원추형 튜브의 측면 아래로 45 ° 각도로 천천히 디스펜스. </li><li> 튜브를 닫고 액체가 관 5-6 시간의 길이를 따라 실행되도록 튜브를 기울여 부드럽게 혼합의. 힘줄, 백색 침전물은 용액 중에 형성 할 수있다. 분 아래에 트립신 시간을 제한합니다. </li><li> 감기 FCS 1ml의 트립신 처리로 반응을 켄칭. 즉시 차가운 PBS 10 ㎖를 첨가하여 희석 FCS. </li><li> 4 ° C에서 5 분 300 XG에 원심 분리 한 후 상층 액을 가만히 따르다과 얼음에 튜브를 반환합니다. </li></ol></li><li> 정착 <ol><li> 경사 분리 후 잔류 체적의 세포를 재현 탁. 필요한 경우, 차가운 PBS 100 μL에 볼륨을 가져. </li><li> 흄 후드에서, 세포에 10 % 중성 완충 포르말린 용액 800 μL를 추가하고 상하 피펫. 튜브는 포르말린 첨가시 따뜻하게. 10 분 동안 37 ℃에서 배양한다. </li></ol></li><li> 투과성으로 <ol><li> 부드러운 흔들림 속도로 벤치 탑 와류 믹서를 설정합니다. </li><li> 10 ㎖의 혈청 학적 피펫으로 추운 (-20 ° C) 메탄올 9 ml에 그립니다. </li><li> 와류 믹서를 통해 세포를 포함하는 튜브를 잡고 부드럽게 흔들리는 시작g 세포. </li><li> 튜브는 여전히 떨고 메탄올을 지속적으로 혼합되는 동안이 세포에 적가 쓰러 튜브에 냉 메탄올을 추가합니다. 메탄올 2ml의 제 후에 분배 속도가 느린 스트림으로 증가 될 수있다. </li><li> 30 분 투과성으로를 완료 할 때 튜브가 얼음에 앉아 보자. 대안 적으로, 세포는 개월까지 -20 ℃에서 저장 될 수있다. </li></ol></li></ol> 의 IgG1과 IgE의 클래스 전환 4. 형광 염색 <ol><li> 원심 분리기에서 5 분 동안 300 XG에 샘플은 메탄올 용액 캔트. 백색 침전물을 세포 펠릿있다. </li><li> 5 분 세포를 씻어 300 XG에서 객실 PBS 1X 온도와 원심 분리기 10 ML을 추가합니다. 한 번 반복합니다. 참고 : 실온 PBS와 펠렛을 세척하는 단계 4.1 이후에 형성 할 수있다 여분의 침전물을 용해. </li><li> 2 % FCS-FACS 완충액 10 mL로 추가 세척을 수행합니다. </li><li> 바닥 판 라운드 96 웰에 샘플을 배포5 분 동안 원심 분리기 (300) XG. 상층 액을 가만히 따르다과 얼음에 접시를 넣어. </li><li> 다음과 같은 항체를 희석 잘 2 % FCS-FACS 버퍼의 60 μL에 얼룩 : 안티 IgM 항체 RPE / Cy7, 1 : 200, 안티의 IgG1 PE, 1 : 600, 안티 IgE에 FITC, 1 : 100, 안티 - CD45R (B220)를 PerCP-Cy5.5, 1 : 200 </li><li> 5 분 염색 한 후, 300 x g에서 5 분 동안 2 % FCS-FACS 완충액 원심 150 μL로 플레이트를 세척한다. </li><li> 5 ML의 FACS 튜브에 40 μm의 나일론 셀 스트레이너를 통해 2 % FCS-FACS 버퍼와 피펫 400 μL에 재현 탁. </li></ol> 5. 다음 게이팅 전략을 사용합니다 : <ol><li> FSC / SSC 플롯 (그림 1)에있는 발파 림프구 인구에서 CD45R (B220) 양성 세포를 분리합니다. </li><li> B220 양성 세포에서 해당 인구를 나타 내기 위해 IgE 항체와의 IgG1 플롯을 사용합니다. </li></ol>

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항 CD40 및 IL-4와 문화에 마우스 비장 B 세포의 자극의 IgG1과 IgE의 5 T 도우미 2 형 (T H 2) 상호 작용을 격려 클래스 전환을 시뮬레이션합니다. B 세포는 비장 세포 총 12 또는 정제 된 비장의 B 세포 (11)의 맥락에서 CSR을 위해 활성화 될 수있다. 프로토콜 (단계 2.3)에서 설명한 바와 같이, B 세포 농축은 선택 사항이며, 그것은 유익 할 것이다 있는지 확인 실험자의 판단이?...

마우스와 인간 면역 글로불린 중쇄 (IGH) 클래스 스위치 재조합 (CSR) 중, 고등학교의는 일정한 지역 엑손 (Cμ가) 삭제 및 다운 스트림 일정한 지역 엑손의 여러 세트 중 하나 (C의 H s의) (예를 들어 Cγ로 대체 μ IG 다른 클래스 (예를 들면 IgG를, IgE 항체, 또는 적 IgA)의 제조에 대한 IgM 항체의 생산에서 발생 Cε 스위치 및 Cα). CSR은 C H의 1의 각 세트 5 &#39;위치 1-10킬로바이트 시퀀스는 스위치 (S) 지역 내에서 발생합니다. 활성화 - 유도 시티 딘 데 아미나 제 (AID) 효소는 시티 딘의 탈 아미노 활동을 통해 CSR을 시작합니다. IgE 항체는 알레르기 질환 2의 주요 중재자 및 IgE의 생산 및 아토피 질환에 대한 새로운 치료 방법으로 문을 열 수 있습니다 규제 방법에 대한 이해이다. 마우스와 인간에서의 IgE는 가장 엄격하게 규제 IG 이소 타입이다. IgE 항체는 일반적으로 팀의 수준 수천에서 감지ES 다른 IGH 미만 3 아이소 타입이지만 높은 질환 상태 4에서 증가 될 수있다. 그러나 IgE 항체에 CSR은 불완전하게 이해된다. B 세포의 체외 활성화는 IL-4 항 CD40 또는 LPS 중 하나와 조합은, 모두의 IgG1과 IgE의 5 CSR을 유도하여. 활성화 된 B 세포를 용해 IgE 항체가 CSR 후 문화 분비 결합 선호도가 낮은의 IgE 수용체 FcεRII / CD23 2,6-을 표현한다. 유동 세포 계측법으로 분석 할 때 따라서, 수용체에 결합 된 IgE의 얼룩 B 세포는 유사 내생 적 IgE 항체 (7)를 발현하는 세포를 B로. 그것은 그 마우스 B를 알려져 있지만 세포는의 IgG1에 클래스 전환의 측정에 방해가 나타나지 않는 우리의 경험에서 선호도가 낮은 IgG의 수용체 FcγRIIB1 (CD32) 8을 표현한다. 배양에서 B 세포 활성화 후 IgE의 전환을 측정하는 경우에는, 표면 - 결합 특이성 IgE 항체는 분석을 가릴 수있다. 공통 염색 방법과 비 IgE 항체 발현 세포가 IgE에 대해 긍정적으로 염색. 여기에 설명 된 마우스 (9)로부터 참의 IgE 발현 B 세포를 CSR 세이를 수행하고 검출하는데 이용 된 전략 - 11. 트립신으로 활성화 된 B 세포의 치료는 일반적인 실험실 시약은 단백질을 소화, 세포 친화 및 막 모두 결합 된 표면의 IgE는 제거합니다. 세포질 IgE에 대한 후속 투과성으로 염색 따라서 진정한 IgE 항체 생산 세포를 보여준다.

<table><tbody><tr><td>Anti-Mouse IgE FITC</td><td>BD Pharmingen</td><td>553415</td><td>clone R35-72</td></tr><tr><td>Rat Anti-Mouse IgG1 PE</td><td>BD Pharmingen</td><td>550083</td><td>clone A85-1</td></tr><tr><td>Methanol</td><td>BDH</td><td>BDH1135-4LP</td><td>Keep at -20 &amp;deg;C</td></tr><tr><td>Falcon Cell Strainer 40 &amp;micro;m Nylon</td><td>Corning</td><td>352340</td><td></td></tr><tr><td>Falcon Cell Strainer 70 &amp;micro;m Nylon</td><td>Corning</td><td>352350</td><td></td></tr><tr><td>Anti-Hu/Mo CD45R(B220) PerCP-Cy5.5</td><td>eBioscience</td><td>45-4052-82</td><td>clone RA3-6B2</td></tr><tr><td>anti-Mouse/Rat CD40</td><td>eBioscience</td><td>16-0402-85</td><td>clone HM40-3</td></tr><tr><td>RPMI Medium 1640</td><td>Gibco</td><td>11875-093</td><td></td></tr><tr><td>HEPES (1M)</td><td>Gibco</td><td>15630-080</td><td></td></tr><tr><td>MEM-NEAA (100x)</td><td>Gibco</td><td>11140-050</td><td></td></tr><tr><td>Phosphate Buffered Saline (10x)</td><td>Lifetechnologies (Corning)</td><td>46-013-CM</td><td></td></tr><tr><td>MACS CD45R (B220) microbeads&amp;nbsp;</td><td>Miltenyi Biotec</td><td>130-049-501</td><td></td></tr><tr><td>MACS purification column</td><td>Miltenyi Biotec</td><td>130-042-401</td><td></td></tr><tr><td>IL-4</td><td>PeproTech</td><td>214-14</td><td>Reconstitute in water or 0.1% BSA</td></tr><tr><td>Formalin Solution, Neutral Buffered, 10%</td><td>Sigma Aldrich</td><td>HT501128-4L</td><td></td></tr><tr><td>Trypsin-EDTA Solution (10x)</td><td>Sigma Aldrich</td><td>T4174-100 ml</td><td>5.0 g Trypsin, 2.0 g EDTA&amp;#8226;4 Na per Liter of 0.9% NaCl</td></tr><tr><td>Penicillin-Streptomycin</td><td>Sigma Aldrich</td><td>P0781-100 ml</td><td>10,000 U Penicillin; 10 mg/ml Streptomycin (100x)</td></tr><tr><td>L-Glutamine 200 mM</td><td>Sigma Aldrich</td><td>G7513</td><td></td></tr><tr><td>2-mercaptoethanol (99%)</td><td>Sigma Aldrich</td><td>M6250-100 ml</td><td></td></tr><tr><td>Red cell lysis buffer</td><td>Sigma Aldrich</td><td>R7757</td><td></td></tr><tr><td>Rat Anti-Mouse IgM RPE/Cy7</td><td>SouthernBiotech</td><td>1140-17</td><td>clone 1B4B1</td></tr><tr><td>HyClone Fetal Calf Serum</td><td>Thermo</td><td>SH30910.03</td><td></td></tr><tr><td>B cell Stimulation media</td><td></td><td></td><td>B cell stimulation media consists of RPMI with fetal calf serum (15%), 20 mM HEPES, 1x MEM-NEAA, 2.0 mM L-glutamine,&amp;nbsp; 1x Penicillin-Streptomycin (Penicillin: 100 U/ml, Streptomycin: 100 &amp;micro;g/ml), Beta-mercaptoethanol (7 &amp;micro;L/L), IL-4 (20 ng/ml), and anti-CD40 (1 &amp;micro;g/ml)</td></tr></tbody></table>

detection of true ige-expressing mouse b lineage cells

B 림프구 면역 글로불린 중쇄 (IGH) 클래스 스위치 재조합 (CSR)은 처음에 IgM 항체 등의 IgA, IgE 항체와 IgG의 다른 고등학교의 이소 타입으로 전환 표현 된 것을 특징으로하는 과정이다. 시험 관내 IGH CSR의 측정은 분자 세포 면역학의 측면 DNA 재조합 및 수리에 이르기까지 생물학적 공정 수의 연구를위한 중요한 방법이다. 관내 CSR 분석은 활성화 된 B 세포에 대한 유세포 측정면 IG 표현식을 수반한다. 의 IgA와 IgG의 서브 클래스의 측정이 간단하지만,이 방법으로 IgE 항체의 측정으로 인해 용해 IgE 항체가 FcεRII에 결합에 문제가있다 / CD23 활성화 B 세포의 표면에 발현. 여기에서 우리는 문화에 CSR을받은 마우스 B 세포를 IgE 항체가 생산의 정확한 측정을위한 고유 한 절차를 설명합니다. 방법 B에 의해 CY 계통 세포의 IgE가 생산의 검출을 허용 세포 표면에 대한 IgE CD23 복합체 트립신 - 매개 절단에 기초toplasmic 염색. 이 절차에 대한 IgE의 CSR 유세포 분석을위한 편리한 해결책을 제공한다.

이 절차가 성공적으로 마우스 B 세포에서 IgE 항체에 CSR을 연구하기 위해 구현되었습니다. 앞서 설명한 바와 같이 11 CSR 측정에서 효율을 입증하기 위해, 우리는 항 CD40 및 IL-4와 마우스 비장 B 세포를 자극. 자극 5 일 후, 세포를 수거하고, 프로토콜 형광 표지 IgM이, IgG1에, IgE 항체, 및 B220 (CD45R) 항체 상술과 염색을 이용하여 처리 하였다. (도 1), + 세포의 IgE에 명확한 인구 깔끔하?...

Watch this Scientific Journal Video about Detection of True IgE-expressing Mouse B Lineage Cells at JoVE.com

Detection of True IgE-expressing Mouse B Lineage Cells

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 ...

detection-of-true-ige-expressing-mouse-b-lineage-cells

Research

JoVE Journal

Immunology and Infection

10.9K 조회수.  Brigham and Women's Hospital and Harvard Medical School. 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.

비디오: Detection of True IgE-expressing Mouse B Lineage 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

Detection and Isolation of Viable Mouse IL-17-Secreting T Cells

The MACS Cytokine Secretion Assay technology allows detection of secreted cytokines on the single cell level and sensitive isolation of viable cytokine-secreting cells. In order to label IL-17-secreting cells, a single cell suspension of mouse splenocytes is prepared and stimulated at 37&deg;C with PMA/ionomycin to induce cytokine secretion. To stop secretion cells are then placed on ice and are exposed to the IL-17 Catch Reagent   a bi-specific antibody that binds to CD45 on the cell surface of leukocytes and to IL-17 as it is secreted and caught near the cell surface. Secretion is then re-started by increasing the temperature to 37&deg;C and IL-17 is trapped by the Catch Reagent. Secretion is then stopped again, by placing cells on ice. To detect the trapped IL-17, cells are incubated with a second IL-17-specific antibody conjugated to biotin and an Anti-Biotin-PE antibody. Cells can now be directly analyzed by flow cytometry or prepared for isolation and enrichment by subsequent labeling with Anti-PE conjugated MicroBeads.

This procedure describes the detection and isolation of mouse TH17 leukocytes that actively secrete IL-17 upon stimulation.

가능한 마우스 IL - 17 - Secreting의 T 세포 탐지 및 격리

The MACS Cytokine Secretion Assay technology allows detection of secreted cytokines on the single cell level and sensitive isolation of viable ...

생물학

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 ...

The bm12 Inducible Model of Systemic Lupus Erythematosus (SLE) in C57BL/6 Mice

Systemic lupus erythematosus (SLE) is an autoimmune disease with diverse clinical and immunological manifestations. Several spontaneous and inducible animal models mirror common components of human disease, including the bm12 transfer model. Upon transfer of bm12 splenocytes or purified CD4 T cells, C57BL/6 mice rapidly develop large frequencies of T follicular helper cells (Tfh), germinal center (GC) B cells, and plasma cells followed by high levels of circulating anti-nuclear antibodies. Since this model utilizes mice on a pure C57BL/6 background, researchers can quickly and easily study disease progression in transgenic or knockout mouse strains in a relatively short period of time. Here we describe protocols for the induction of the model and the quantitation Tfh, GC B cells, and plasma cells by multi-color flow cytometry. Importantly, these protocols can also be used to characterize disease in most mouse models of SLE and identify Tfh, GC B cells, and plasma cells in other disease models.

The bm12 Inducible Model of Systemic Lupus Erythematosus SLE in C57BL/6 Mice

The transfer of bm12 lymphocytes into a C57BL/6 recipient is an established model of systemic lupus erythematosus. Here we describe how to initiate disease using this model and how to characterize T follicular helper cells, germinal center B cells and plasma cells by flow cytometry.

C57BL / 6 마​​우스의 전신 홍 반성 루푸스의 BM12 유도 성 모델 (SLE)

Systemic lupus erythematosus (SLE) is an autoimmune disease with diverse clinical and immunological manifestations. Several spontaneous and inducible ...

의학

Identification and Analysis of Mouse Erythroid Progenitors using the CD71/TER119 Flow-cytometric Assay

The study of erythropoiesis aims to understand how red cells are formed from earlier hematopoietic and erythroid progenitors. Specifically, the rate of red cell formation is regulated by the hormone erythropoietin (Epo), whose synthesis is triggered by tissue hypoxia. A threat to adequate tissue oxygenation results in a rapid increase in Epo, driving an increase in erythropoietic rate, a process known as the erythropoietic stress response. The resulting increase in the number of circulating red cells improves tissue oxygen delivery. An efficient erythropoietic stress response is therefore critical to the survival and recovery from physiological and pathological conditions such as high altitude, anemia, hemorrhage, chemotherapy or stem cell transplantation. 
The mouse is a key model for the study of erythropoiesis and its stress response. Mouse definitive (adult-type) erythropoiesis takes place in the fetal liver between embryonic days 12.5 and 15.5, in the neonatal spleen, and in adult spleen and bone marrow. Classical methods of identifying erythroid progenitors in tissue rely on the ability of these cells to give rise to red cell colonies when plated in Epo-containing semi-solid media. Their erythroid precursor progeny are identified based on morphological criteria. Neither of these classical methods allow access to large numbers of differentiation-stage-specific erythroid cells for molecular study. Here we present a flow-cytometric method of identifying and studying differentiation-stage-specific erythroid progenitors and precursors, directly in the context of freshly isolated mouse tissue. The assay relies on the cell-surface markers CD71, Ter119, and on the flow-cytometric 'forward-scatter' parameter, which is a function of cell size. The CD71/Ter119 assay can be used to study erythroid progenitors during their response to erythropoietic stress in vivo, for example, in anemic mice or mice housed in low oxygen conditions. It may also be used to study erythroid progenitors directly in the tissues of genetically modified adult mice or embryos, in order to assess the specific role of the modified molecular pathway in erythropoiesis.

A flow-cytometric method for identification and molecular analysis of differentiation-stage-specific murine erythroid progenitors and precursors, directly in freshly &#x2013;harvested mouse bone marrow, spleen or fetal liver. The assay relies on cell-surface markers CD71, Ter119, and cell size.

CD71/TER119 흐름 cytometric 어세를 사용하여 마우스 Erythroid Progenitors의 제품 식별 및 분석

The study of erythropoiesis aims to understand how red cells are formed from earlier hematopoietic and erythroid progenitors. Specifically, the rate of ...

Characterization of Thymus-dependent and Thymus-independent Immunoglobulin Isotype Responses in Mice Using Enzyme-linked Immunosorbent Assay

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a formidable defense against invading pathogens via diverse mechanisms. One major goal of vaccination is to induce protective antigen-specific antibodies to prevent life-threatening infections. Both thymus-dependent (TD) and thymus-independent (TI) antigens can elicit robust antigen-specific IgM responses and can also induce the production of isotype-switched antibodies (IgG, IgA and IgE) as well as the generation of memory B cells with the help provided by antigen presenting cells (APCs). Here, we describe a protocol to characterize TD and TI Ig isotype responses in mice using enzyme-linked immunosorbent assay (ELISA). In this protocol, TD and TI Ig responses are elicited in mice by intraperitoneal (i.p.) immunization with hapten-conjugated model antigens TNP-KLH (in alum) and TNP-polysaccharide (in PBS), respectively. To induce TD memory response, a booster immunization of TNP-KLH in alum is given at 3 weeks after the first immunization with the same antigen/adjuvant. Mouse sera are harvested at different time points before and after immunization. Total serum Ig levels and TNP-specific antibodies are subsequently quantified using Ig isotype-specific Sandwich and indirect ELISA, respectively. In order to correctly quantify the serum concentration of each Ig isotype, the samples need to be appropriately diluted to fit within the linear range of the standard curves. Using this protocol, we have consistently obtained reliable results with high specificity and sensitivity. When used in combination with other complementary methods such as flow cytometry, in vitro culture of splenic B cells and immunohistochemical staining (IHC), this protocol will allow researchers to gain a comprehensive understanding of antibody responses in a given experimental setting.

In this paper, we describe a protocol to characterize T-dependent and T-independent immunoglobulin (Ig) isotype responses in mice using ELISA. This method used alone or in combination with flow cytometry will allow researchers to identify differences in B cell-mediated Ig isotype responses in mice following T-dependent and T-independent antigen immunization.

Thymus 종속 및 독립 Thymus 면역 글로불린 Isotype 반응 효소 연결 된 Immunosorbent 분석 결과 사용 하 여 마우스의 특성

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a ...

Retroviral Overexpression of CXCR4 on Murine B-1a Cells and Adoptive Transfer for Targeted B-1a Cell Migration to the Bone Marrow and IgM Production

As cell function is influenced by niche-specific factors in the cellular microenvironment, methods to dissect cell localization and migration can provide further insight on cell function. B-1a cells are a unique B cell subset in mice that produce protective natural IgM antibodies against oxidation-specific epitopes that arise during health and disease. B-1a cell IgM production differs depending on B-1a cell location, and therefore it becomes useful from a therapeutic standpoint to target B-1a localization to niches supportive of high antibody production. Here we describe a method to target B-1a cell migration to the bone marrow by retroviral-mediated overexpression of the C-X-C motif chemokine receptor 4 (CXCR4). Gene induction in primary murine B cells can be challenging and typically yields low transfection efficiencies of 10-20% depending on technique. Here we demonstrate that retroviral transduction of primary murine B-1a cells results in 30-40% transduction efficiency. This method utilizes adoptive cell transfer of transduced B-1a cells into B cell-deficient recipient mice so that donor B-1a cell migration and localization can be visualized. This protocol can be modified for other retroviral constructs and can be used in diverse functional assays post-adoptive transfer, including analysis of donor cell or host cell phenotype and function, or analysis of soluble factors secreted post B-1a cell transfer. The use of distinct donor and recipient mice differentiated by CD45.1 and CD45.2 allotype and the presence of a GFP reporter within the retroviral plasmid could also enable detection of donor cells in other, immune-sufficient mouse models containing endogenous B cell populations.

Here we describe a method for retroviral overexpression and adoptive transfer of murine B-1a cells to examine in vivo B-1a cell migration and localization. This protocol can be extended for diverse downstream functional assays including quantification of donor B-1a cell localization or analysis of donor cell-derived secreted factors post-adoptive transfer.

뮤린 B-1a 세포에 CXCR4의 레트로 바이러스 과발현 및 골수와 IgM 생산에 표적 B-1a 세포 이동을 위한 채택 전송

As cell function is influenced by niche-specific factors in the cellular microenvironment, methods to dissect cell localization and migration can provide ...

Flow Cytometric Characterization of Murine B Cell Development

Extensive studies have characterized the development and differentiation of murine B cells in secondary lymphoid organs. Antibodies secreted by B cells have been isolated and developed into well-established therapeutics. Validation of murine B cell development, in the context of autoimmune prone mice, or in mice with modified immune systems, is a crucial component of developing or testing therapeutic agents in mice and is an appropriate use of flow cytometry. Well established B cell flow cytometric parameters can be used to evaluate B cell development in the murine peritoneum, bone marrow, and spleen, but a number of best practices must be adhered to. In addition, flow cytometric analysis of B cell compartments should also complement additional readouts of B cell development. Data generated using this technique can further our understanding of wild type, autoimmune prone mouse models as well as humanized mice that can be used to generate antibody or antibody-like molecules as therapeutics.

We describe herein a simple analysis of the heterogeneity of the murine immune B cell compartment in the peritoneum, spleen, and bone marrow tissues by flow cytometry. The protocol can be adapted and extended to other mouse tissues.

뮤린 B 세포 발달의 유동 세포 측정 특성화

Extensive studies have characterized the development and differentiation of murine B cells in secondary lymphoid organs. Antibodies secreted by B cells ...

Flow Cytometric Analysis for Identification of the Innate and Adaptive Immune Cells of Murine Lung

The respiratory tract is in direct contact with the outside environment and requires a precisely regulated immune system to provide protection while suppressing unwanted reactions to environmental antigens. Lungs host several populations of innate and adaptive immune cells that provide immune surveillance but also mediate protective immune responses. These cells, which keep the healthy pulmonary immune system in balance, also participate in several pathological conditions such as asthma, infections, autoimmune diseases, and cancer. Selective expression of surface and intracellular proteins provides unique immunophenotypic properties to the immune cells of the lung. Consequently, flow cytometry has an instrumental role in the identification of such cell populations during steady-state and pathological conditions. This paper presents a protocol that describes a consistent and reproducible method to identify the immune cells that reside in the lungs of healthy mice under steady-state conditions. However, this protocol can also be used to identify changes in these cell populations in various disease models to help identify disease-specific changes in the lung immune landscape.

In this study, we present an effective and reproducible protocol to isolate the immune populations of the murine respiratory system. We also provide a method for the identification of all innate and adaptive immune cells that reside in the lungs of healthy mice, using a 9-color-based flow cytometry panel.

뮤린 폐의 선천적 및 적응성 면역 세포의 확인을 위한 유세포 분석

The respiratory tract is in direct contact with the outside environment and requires a precisely regulated immune system to provide protection while ...

Isolation of Group 2 Innate Lymphoid Cells from Mouse Nasal Mucosa to Detect the Expression of CD226

With abundant research on group 2 innate lymphoid cells (ILC2s) published over the years, ILC2s are widely known to be implicated in regulating various pathological processes, including anti-helminth immunity, tissue repair, thermogenesis, and autoimmune diseases such as asthma and allergic rhinitis (AR). ILC2s permanently reside in peripheral tissues such as the skin, gut, lungs, and nasal cavity; however, there is limited information about their exact functions in nasal mucosal immunity. CD226 is an activating costimulatory molecule, mainly expressed on natural killer (NK) cells, T cells, and inflammatory monocytes. However, whether ILC2s express CD226 or play a role in the pathogenesis of ILC2s-related diseases remains unknown. Here, we established a method to isolate and identify ILC2s from the nasal mucosa and detected CD226 expression on ILC2s obtained from healthy and AR mice. Herein, we describe this protocol for the isolation and identification of ILC2s from mouse nasal mucosa, which will help explore the internal pathological mechanism of immunological disorders in nasal mucosal diseases.

Group 2 innate lymphoid cells (ILC2s), implicated in type 2 inflammation, mainly participate in response to helminth infection, allergic diseases, metabolic homeostasis, and tissue repair. In this study, a procedure to isolate ILC2s from murine nasal mucosa and detect the expression of CD226 is demonstrated.

CD226의 발현을 검출하기 위해 마우스 비강 점막에서 그룹 2 선천성 림프 세포 분리

With abundant research on group 2 innate lymphoid cells (ILC2s) published over the years, ILC2s are widely known to be implicated in regulating various ...

진정한 검출 마우스 B 리니지 세포를 IgE 항체가 발현

요약

더 많은 비디오 탐색

이 비디오의 챕터

Murine B 세포를 정화에 유도 및 클래스 스위치 재조합 평가

가능한 마우스 IL - 17 - Secreting의 T 세포 탐지 및 격리

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

C57BL / 6 마우스의 전신 홍 반성 루푸스의 BM12 유도 성 모델 (SLE)

CD71/TER119 흐름 cytometric 어세를 사용하여 마우스 Erythroid Progenitors의 제품 식별 및 분석

Thymus 종속 및 독립 Thymus 면역 글로불린 Isotype 반응 효소 연결 된 Immunosorbent 분석 결과 사용 하 여 마우스의 특성

뮤린 B-1a 세포에 CXCR4의 레트로 바이러스 과발현 및 골수와 IgM 생산에 표적 B-1a 세포 이동을 위한 채택 전송

뮤린 B 세포 발달의 유동 세포 측정 특성화

뮤린 폐의 선천적 및 적응성 면역 세포의 확인을 위한 유세포 분석

CD226의 발현을 검출하기 위해 마우스 비강 점막에서 그룹 2 선천성 림프 세포 분리

진정한 검출 마우스 B 리니지 세포를 IgE 항체가 발현

요약

더 많은 비디오 탐색

이 비디오의 챕터

Murine B 세포를 정화에 유도 및 클래스 스위치 재조합 평가

가능한 마우스 IL - 17 - Secreting의 T 세포 탐지 및 격리

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

C57BL / 6 마​​우스의 전신 홍 반성 루푸스의 BM12 유도 성 모델 (SLE)

CD71/TER119 흐름 cytometric 어세를 사용하여 마우스 Erythroid Progenitors의 제품 식별 및 분석

Thymus 종속 및 독립 Thymus 면역 글로불린 Isotype 반응 효소 연결 된 Immunosorbent 분석 결과 사용 하 여 마우스의 특성

뮤린 B-1a 세포에 CXCR4의 레트로 바이러스 과발현 및 골수와 IgM 생산에 표적 B-1a 세포 이동을 위한 채택 전송

뮤린 B 세포 발달의 유동 세포 측정 특성화

뮤린 폐의 선천적 및 적응성 면역 세포의 확인을 위한 유세포 분석

CD226의 발현을 검출하기 위해 마우스 비강 점막에서 그룹 2 선천성 림프 세포 분리

C57BL / 6 마우스의 전신 홍 반성 루푸스의 BM12 유도 성 모델 (SLE)