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 border="0" cellpadding="0" cellspacing="0" width="958">
	<tbody>
		<tr height="37">
			<td height="37" style="height:37px;width:301px;">Name</td>
			<td style="width:179px;">Company</td>
			<td style="width:135px;">Catalog Number</td>
			<td style="width:343px;">Comments</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Anti-Mouse IgE FITC</td>
			<td>BD Pharmingen</td>
			<td>553415</td>
			<td>clone R35-72</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Rat Anti-Mouse IgG1 PE</td>
			<td>BD Pharmingen</td>
			<td>550083</td>
			<td>clone A85-1</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Methanol</td>
			<td>BDH</td>
			<td>BDH1135-4LP</td>
			<td>Keep at -20 &#176;C</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Falcon Cell Strainer 40 &#181;m Nylon</td>
			<td>Corning</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Falcon Cell Strainer 70 &#181;m Nylon</td>
			<td>Corning</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Anti-Hu/Mo CD45R(B220) PerCP-Cy5.5</td>
			<td>eBioscience</td>
			<td>45-4052-82</td>
			<td>clone RA3-6B2</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">anti-Mouse/Rat CD40</td>
			<td>eBioscience</td>
			<td>16-0402-85</td>
			<td>clone HM40-3</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">RPMI Medium 1640</td>
			<td>Gibco</td>
			<td>11875-093</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">HEPES (1M)</td>
			<td>Gibco</td>
			<td>15630-080</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">MEM-NEAA (100X)</td>
			<td>Gibco</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Phosphate Buffered Saline (10X)</td>
			<td>Lifetechnologies (Corning)</td>
			<td>46-013-CM</td>
			<td></td>
		</tr>
		<tr height="66">
			<td height="66" style="height:67px;">MACS CD45R (B220) microbeads&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>130-049-501</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">MACS purification column</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">IL-4</td>
			<td>PeproTech</td>
			<td>214-14</td>
			<td>Reconstitute in water or 0.1% BSA</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Formalin Solution, Neutral Buffered, 10%</td>
			<td>Sigma Aldrich</td>
			<td>HT501128-4L</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Trypsin-EDTA Solution (10X)</td>
			<td>Sigma Aldrich</td>
			<td>T4174-100mL</td>
			<td>5.0g Trypsin, 2.0g EDTA&#376;4Na per Liter of 0.9% NaCl</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Penicillin-Streptomycin</td>
			<td>Sigma Aldrich</td>
			<td>P0781-100mL</td>
			<td>10,000U Penicillin; 10 mg/mL Streptomycin (100X)</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">L-Glutamine 200mM</td>
			<td>Sigma Aldrich</td>
			<td>G7513</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">2-mercaptoethanol (99%)</td>
			<td>Sigma Aldrich</td>
			<td>M6250-100mL</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Red cell lysis buffer</td>
			<td>Sigma Aldrich</td>
			<td>R7757</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Rat Anti-Mouse IgM RPE/cy7</td>
			<td>SouthernBiotech</td>
			<td>1140-17</td>
			<td>clone 1B4B1</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">HyClone Fetal Calf Serum</td>
			<td>Thermo</td>
			<td>SH30910.03</td>
			<td></td>
		</tr>
		<tr height="161">
			<td height="161" style="height:161px;">B cell Stimulation media</td>
			<td></td>
			<td></td>
			<td style="width:343px;">B cell stimulation media consists of RPMI with fetal calf serum (15%), 20 mM HEPES, 1X MEM-NEAA, 2.0mM L-glutamine,&#160; 1X Penicillin-Streptomycin (Penicillin:100 U/ml, Streptomycin: 100 &#181;g/ml), Beta-mercaptoethanol (7 &#181;L/L), IL-4 (20 ng/mL), and anti-CD40 (1 &#181;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

10.8K 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

Murine Model of CD40-activation of B cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients1-4. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro5. Furthermore they efficiently take up, process and present antigens to T cells6,7. CD40-B cells were shown to not only prime na&iacute;ve, but also expand memory T cells8,9. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa)11. Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (&gt;90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach12.

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

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 ...

연구

면역학 및 감염병학

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 ...

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 ...

Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency and type of chromosome aberrations in different cell types allows defects in DNA repair pathways to be elucidated. Understanding mammalian DNA repair biology has been greatly helped by the production of mice with knockouts in specific genes. The goal of this protocol is to quantify genomic instability in mouse B lymphocytes. Labeling of the telomeres using PNA-FISH probes (peptide nucleic acid - fluorescent in situ hybridization) facilitates the rapid analysis of genomic instability in metaphase chromosome spreads. B cells have specific advantages relative to fibroblasts, because they have normal ploidy and a higher mitotic index. Short-term culture of B cells therefore enables precise measurement of genomic instability in a primary cell population which is likely to have fewer secondary genetic mutations than what is typically found in transformed fibroblasts or patient cell lines.

DNA repair pathways are essential for maintenance of genomic integrity and preventing mutation and cancer. The goal of this protocol is to quantify genomic instability by direct observation of chromosome aberrations in metaphase spreads from mouse B cells using fluorescent in situ hybridization (FISH) for telomeric DNA repeats.

PNA-FISH에 의해 마우스 B 림프구에서 염색체 이상을 신속하게 분석

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency ...

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, ...

Rapid Molecular Detection and Differentiation of Influenza Viruses A and B

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality every year. The Influenza A and B assay was the first CLIA-waived molecular rapid flu test available. The Influenza A and B test works by employing isothermal amplification with influenza-specific primers followed by target detection with molecular beacon probes. Here, the performance of the Influenza A and B assay on frozen, archived nasopharyngeal swab (NPS) specimens stored in viral transport medium (VTM) were compared to a respiratory panel assay.
The performance of the Influenza A and B assay was evaluated by comparing the results to the respiratory panel reference method. The sensitivity for total influenza virus A was 67.5% (95% CI (CI), 56.6-78.5) and the specificity was 86.9% (CI, 71.0-100). For influenza virus B testing, the sensitivity and specificity were 90.2% (CI, 68.5-100) and 98.8% (CI, 68.5-100), respectively.
This system has the advantage of a significantly shorter test time than any other currently available molecular assay and the simple, pipette-free procedure runs on a fully integrated, closed, small-footprint system. Overall, the Influenza A and B assay evaluated in this study has the potential to serve as a point-of-care rapid influenza diagnostic test.

We describe a rapid, molecular-based Influenza A and B assay. The Influenza assay detects each target within 15 min by employing isothermal amplification with influenza-specific primers followed by target detection with molecular beacon probes. The Influenza A and B assay is user-friendly and required minimal hands-on time to perform.

빠른 분자 탐지 및 인플루엔자 바이러스 A와 B의 차별화

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality ...

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 ...

Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote inflammatory and autoinflammatory diseases. Inflammasomes are activated by cytosolic pattern recognition receptors, including members of the NOD-like receptor (NLR) family. These receptors oligomerize upon the detection of microbial or damage-associated stimuli. Subsequent recruitment of the adaptor protein ASC forms a microscopically visible inflammasome complex, which activates caspase-1 through proximity-induced auto-activation. Following the activation, caspase-1 cleaves pro-IL-1&#946; and pro-IL-18, leading to the activation and secretion of these pro-inflammatory cytokines. Caspase-1 also mediates the inflammatory form of cell death termed pyroptosis, which features the loss of membrane integrity and cell lysis. Caspase-1 cleaves gasdermin D, releasing the N-terminal fragment which forms plasma membrane pores, leading to osmotic lysis.
In vitro, the activation of caspase-1 can be determined by labeling bone marrow-derived macrophages with the caspase-1 activity probe FAM-YVAD-FMK and by labeling the cells with antibodies against the adaptor protein ASC. This technique allows the identification of inflammasome formation and caspase-1 activation in individual cells using fluorescence microscopy. Pyroptotic cell death can be detected by measuring the release of cytosolic lactate dehydrogenase into the medium. This procedure is simple, cost effective and performed in a 96-well plate format, allowing adaptation for screening. In this manuscript, we show that activation of the NLRP3 inflammasome by nigericin leads to the co-localization of the adaptor protein ASC and active caspase-1, leading to pyroptosis.

We describe the detection of NLRP3 inflammasome activation on cellular basis using fluorescence microscopy and staining for active caspase-1 and the adaptor, ASC. A lactate dehydrogenase release assay is presented to detect pyroptotic lysis on a population basis. These techniques can be adapted to study many aspects of inflammasome biology.

Inflammasome 활성화 및 Pyroptotic 세포 죽음 Murine 골 수 유래 세포에서의 검출

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote ...

Identification and Isolation of Oligopotent and Lineage-committed Myeloid Progenitors from Mouse Bone Marrow

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for hematological and immunological analyses. For example, studies of the cellular and molecular properties of myeloid progenitor populations can reveal mechanisms underlying leukemic transformation, or demonstrate how the immune system responds to pathogen exposure. Previously described flow cytometry strategies for myeloid progenitor identification have enabled significant advances in many fields, but the fractions they identify are very heterogeneous. The most commonly used gating strategies define bone marrow fractions that are enriched for the desired populations, but also contain large numbers of &#34;contaminating&#34; progenitors. Our recent studies have resolved much of this heterogeneity, and the protocol we present here permits the isolation of 6 subpopulations of oligopotent and lineage-committed myeloid progenitors from 2 previously described bone marrow fractions. The protocol describes 3 stages: 1) isolation of bone marrow cells, 2) enrichment for hematopoietic progenitors by magnetic-activated cell sorting (lineage depletion by MACS), and 3) identification of myeloid progenitor subsets by flow cytometry (including fluorescence-activated cell sorting, FACS, if desired). This approach permits progenitor quantification and isolation for a variety of in vitro and in vivo applications, and has already yielded novel insight into pathways and mechanisms of neutrophil, monocyte, and DC differentiation.

We demonstrate how to identify and isolate 6 subsets of myeloid progenitors from murine bone marrow using a combination of magnetic and fluorescence sorting (MACS and FACS). This protocol can be used for in vitro culture assays (methylcellulose or liquid cultures), in vivo adoptive transfer experiments, and RNA/protein analyses.

식별 및 Oligopotent 및 계보 커밋된 골수성 창시자 마우스 골 수에서 격리

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for ...

Detection of Low Copy Number Integrated Viral DNA Formed by In Vitro Hepatitis B Infection

Hepatitis B Virus (HBV) is a common blood-borne pathogen causing liver cancer and liver cirrhosis resulting from chronic infection. The virus generally replicates through an episomal DNA intermediate; however, integration of HBV DNA fragments into the host genome has been observed, even though this form is not necessary for viral replication. The exact purpose, timing, and mechanism(s) by which HBV DNA integration occurs is not yet clear, but recent data show that it occurs very early after infection. Here, the in vitro generation and detection of HBV DNA integrations are described in detail. Our protocol specifically amplifies single copies of virus-cell DNA integrations and allows both absolute quantification and single-base pair resolution of the junction sequence. This technique has been applied to various HBV-susceptible cell types (including primary human hepatocytes), to various HBV mutants, and in conjunction with various drug exposures. We foresee this technique becoming a key assay to determine the underlying molecular mechanisms of this clinically relevant phenomenon.

Detection of Low Copy Number Integrated Viral DNA Formed by In Vitro Hepatitis B Infection

We describe here the in vitro generation of HBV DNA via a Hepatitis B virus infection system and the highly sensitive detection of its (1&#8211;2 copies) integration using inverse nested PCR.

낮은 복사의 번호 통합된 바이러스 성 DNA 생체 외에서 B 형 간염 감염에 의해 형성 된

Hepatitis B Virus (HBV) is a common blood-borne pathogen causing liver cancer and liver cirrhosis resulting from chronic infection. The virus generally ...