The authors would like to acknowledge Dr. Ellen M. Fox for her work regarding this model of local anaphylaxis. This work was supported by the Uniformed Services University of the Health Sciences grant number R073UE.

1 민감 마우스 <ol><li> 20 ㎎ / ㎖의 최종 농도로 1X PBS에 희석하여 OVA OVA 스톡 용액을 준비한다. 향후 사용을 위해 -80 ° C에서 cryovials 및 저장소에 분주을 동결. </li><li> 실온으로 20 ㎎ / ㎖ OVA 스톡 하나 이상의 분취 량을 준비한다. 모자 5 ㎖ 둥근 바닥 폴리스티렌 튜브에 1 ㎎ / ㎖의 최종 농도로 1X PBS에 희석 OVA. 소용돌이 간략하게 혼합한다. </li><li> : 1 비율 중속 설정 와류에 1 ㎎ / ㎖ OVA 함유 폴리스티렌 튜브 채 하나를 생성하기 위해 상기 튜브를 와동 계속하면서, 완전히 균질 Imject 명반 (40 ㎎ / ㎖)을 적하의 동량을 추가 면역원에 명반. </li><li> 소용돌이에 튜브 및 테이프 튜브에 뚜껑을 놓습니다. 낮은 설정에 소용돌이를 켜고 OVA가 30 분 동안 졸업생을 흡수 할 수 있습니다. <ol><li> 시간 많은 양의 OVA / 졸업생을 준비하고 쥐를 민감 사이를 통과하는 것을 허용하지 마십시오. 과민 반응은 OVA / 졸업생 후에는 준비를 직접 완료 할 수없는 경우상온에서 이온, 저장 OVA / 명반. 사용하기 전에 1 분 동안 소용돌이. </li></ol></li><li> OVA / 졸업생 혼합물과 함께 1 ml의 인슐린 주사기를로드합니다. 27 게이지 바늘과 주사기를 씌우고. </li><li> OVA의 50 μg 및 졸업생의 2 mg을 마우스 당 복용량 BALB / c 마우스의 복막에 OVA / 졸업생의 100 μl를 주입한다. </li><li> 음성 대조군의 경우, PBS에 쥐의 또 다른 그룹을 민감. OVA를 생략, 위에 나열된와 동일한 방식으로 졸업생과 PBS의 1 : 1 혼합물 일을 준비합니다. </li><li> 3 주사 총 삼주에 대한 일주일에 한 번 모든 마우스 (OVA와 PBS 제어) 민감. 최종 주입 한 후, 지역 아나필락시스 분석을 수행하기 전에 적어도 이주를 기다립니다. </li></ol> 2 지역 아나필락시스 분석 <ol><li> 실내 온도 OVA 원액을 가져와. (: 1X PBS에 희석 OVA 4 1) 1.5 ㎖의 마이크로 원심 튜브에, PBS 5 ㎎ / ㎖의 최종 농도 20 ㎎ / ㎖ OVA 스톡을 희석한다. 소용돌이 간략하게 혼합한다. </li><li> RC 2 &lt;사용하여 마우스를 마취/ SUP&gt; 설치류 마취 시스템 (또는 동등한 기화 전달 시스템). 이소 플루 란과 저수지를 채우고 O 2 공기 흐름을 켭니다. 유도 챔버에 넣어 마우스와는 마취를 시작하는 5 % 이소 플루 란 조절 다이얼을 조절합니다. 생쥐가 완전히 마취되면, 이동성의 부족에 의해 지시 깊은 마취를 유지하도록 3 % 이소 플루 란으로 제어 다이얼을 조정한다. 이 절차에서 마취의 목적은 동물을 고정화하는 것이다. 귀와 꼬리 정맥 주사는 동물에 심각한 통증이나 고통을 유발하지 않습니다. 참고 : 아이소 플루 란 인간의 생식 시스템에 부정적인 영향을 미칠 수 있습니다. 배기 가스를 중화 환기가 잘되는 방에서 사용하고 유도 챔버에 청소 통을 첨부해야합니다. 이소 플루 란을 사용하는 동안 임신 한 경우 노출을 최소화하기 위해 카본 필터 마스크를 착용하십시오. </li><li> 로드 31 G 바늘로 덮인 10.3 cc의 인슐린 주사기로 5 ㎎ / ㎖ OVA의 10 μL. 10 μL와 또 다른 10.3 cc의 인슐린 주사기를로드의 1X PBS. </li><li> 해부 현미경을 사용하여, OVA의 50 μg의 도전 복용량 마취 마우스 오른쪽 귀에 OVA의 10 μl를 주입. 기타 용량은 같은 20 μg으로도 허용됩니다. <ol><li> 15 ML 원뿔 튜브 주위에 스카치 테이프 (접착면을 위로) 포장, 사출에 귀를 안정화합니다. 테이프에 오른쪽 손잡이의 등 쪽을 고정합니다. 귀의 두 잎 사이에 바늘 베벨 쪽을 삽입합니다. 천천히 모든 혈관을 칠 않도록주의하면서 귀 조직의 중심으로 OVA를 주입. </li></ol></li><li> 상술 한 바와 같은 방법을 사용하여, 음성 대조군으로서 왼쪽 귀에 10 ㎕의 PBS를 주입. </li><li> 3 분을 기다립니다. <ol><li> 이 시간 동안, 저 지부에 마우스를 위치시키고 1 분 혈관을 팽창하기위한 열 램프 하에서 나 온수 욕에서 꼬리를 잡아. </li></ol></li><li> 천천히 꼬리 V로 0.5 % 에반스 블루 염료의 200 μl를 주입, 27 G 바늘로 덮인 1 ml의 인슐린 주사기를 사용하여마우스의 EIN. 전체 200 μL 전에 용기 구조를 파괴 할 수있다 급속 주사 투여된다. 이 경우, 꼬리의 반대측에 정맥에 염료의 잔량을 주입하도록 시도한다. </li><li> 10 분을 기다립니다. </li><li> 마우스를 안락사와 집게 및 수술 가위를 사용하여 두 귀를 제거합니다. <ol><li> 어떠한 압력이 귀 중심에 주사 부위에 적용되지 않는 것을 확인하고, 겸자와 귀의 가장자리를 잡아. 주변 절단함으로써 최대한 귀 조직을 제거 아니지만 통해 귀 연골 기지 존재 릿지. 모피의 소량 절제 귀 존재할 수있다; 이 분석에 영향을 미치지 않습니다. </li></ol></li><li> 나누어지는 1.5 ML의 microcentrifuge 튜브로 포름 아미드 700 μL. 귀 당 하나의 튜브 (마우스 당 두 개의 튜브)를 준비합니다. 참고 : 포름 아미드가, 기형 돌연변이, 및 자극이다. 장갑, 보안경 및 안면 보호구를 착용 할 것. 취급시 임산부는주의해야합니다의 포름 아미드가 태아 독성이 될 수 있습니다. </li><li> , 포름 아미드 튜브를 모자, 그리고 63 ° C로 설정 건열 화장실에서 하룻밤 배양 귀를 추가합니다. 귀는 여전히 배양 후에 약간 푸른 나타날 수 있습니다; 그러나 염료의 대부분 포름으로 추출한다. </li><li> 96 웰 플레이트에, 중복, 각 샘플의 300 μl를 추가합니다. 이 샘플에 존재하는 경우 모피 전사 피한다. 우물에 존재하는 기포가 없는지 그들은 흡광도 판독에 영향을 미칠 것으로 확인합니다. </li><li> 96 웰 플레이트에, 중복, 포름 아미드 300 μl를 추가합니다. 이 우물은 공백으로 사용됩니다. </li><li> 620 nm에서 흡광도를 측정하는 분광 광도계를 사용한다. 각 샘플 독서에서 빼기 포름 아미드 우물. 참고 : 모든 실험 군인 대학 기관 동물 관리 및 사용위원회에 의해 승인 된 프로토콜에 따라 수행되었다. </li></ol>

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The authors have nothing to disclose.

알레르기 질환의 많은 마커기도 도전의 설정에서 기관지 유체에 순환 히스타민의 Th2 사이토 카인의 생산, 세포 모집의 수준의 변화를 포함하여 알레르기 항원의 반칙으로 알레르기 반응의 강도를 평가하는 데 사용됩니다. 이러한 매개 변수에 변화를 모니터링하는 알레르기 반응을 연구하는 것도 중요하지만, 생물학적 마커는 항상 임상 알레르기 질환과 상관 관계가 없습니다. 이 프로토콜에 설?...

타입 I의 과민 반응은 비만 세포와 호염기구의 표면에 IgE 항체의 항원 - 유도 된 가교 결합에 의해 매개된다. 이 세포의 탈과립과 히스타민, 트립 타제 및 혈소판 활성화 인자 (2)와 같은 혈관 작용 및 염증 매개 물질의 방출에 발생합니다. 탈과립 동안 예비 성형 매개 물질의 방출에 이어, 비만 세포는 합성하고 또한 혈관 투과성 3을 증가하는 프로스타글란딘과 류코트리엔을 놓습니다. 초기 임상 반응이 급격히 발생하여 &quot;즉각적인 반응&quot;이라한다. 피부에, 피부 발진 및 플레어 반응은 항원 도전 분 이내에 쉽게 볼 수 있습니다. 도전 복용량에 따라, &quot;늦은 위상 응답&quot;몇 시간 후를 관찰하는 것이 가능하다. 후기의 부종은 조직이로 현지화 부종 및 백혈구 모집 때문이다. 히스타민, 일반적에 참여 주요 매개체로 간주즉각적인 알레르기 반응은, 한 (HR1)의 선박과 히스타민 수용체 2 (HR2) 평활근에 표현에 표현 된 히스타민 수용체에 작용한다. 결합 된 효과는 염증 넷의 사이트에서 혈액의 흐름과 혈관 투과성을 증가시킨다. 알레르기 동물 모델의 다양한 알레르기 성 천식, 전신성 아나필락시스, 지역 과민증의 모델을 포함한 알러지 염증과 관련된 기전을 연구하기 위해 개발되었다. 정맥 염료 관리는 출판물이 1920 년대 5로 다시이 기술 데이트를 기술로, 거의 한 세기에 대 한 동물 모델에서 지역화 된 알레르기 반응을 측정하는 데 사용되었다. 토끼, 기니피그 즉각적인 과민성 반응을 검사하는 데 사용되는 제 동물 모델이었으며, 가장 민감한 반응은 일반적으로 귀 5,6에서 발견되었다. 분석은 이후 래트 (7) 및 마우스 (8)에 사용하기 위해 유효 하였다. 역사적으로,실험 다양한 방법 전에 염료를 주입, 염료를 주입하기 전에 항원의 주입에 항원을 주입하고, 염료 및 항원의 동시 분사로 사용되었다. 알레르기 반응을 측정하기위한 수단으로서 염료 정맥 투여는 수동, 활성 측정에 사용하고, 수동적 반응 5,9 반전 될 수있는 다목적 분석이다. 수많은 염료 트리 판 블루, Pontamine 스카이 블루, 에반스 블루,이기 블루 536, 인도 잉크 5,6,9 포함 알레르기 반응을 평가하기 위해 사용되었다. 0.5 % 에반스 블루의 용액은 현재 피부에 알레르기 반응을 측정하기 위해 사용되는 표준 염료이다. 도전에 대한 아나필락시스 반응은 과도하다; 최대 강도 (10) 내에 도달 - 염료 주입 15 분, 염료에 관계없이 구에 사용되는 동물 종, 공격 후 30 분을 투여하는 경우에는 반응은 볼 수 없습니다. 염료의 혈관 외 유출의 정량화는 원래이었다LY 파란색 염료 7-9으로 표시된 바와 같이 팽진 크기를 측정하여 얻을 수 있습니다. 또한 degranulated 비만 세포의 수는 반응 부위에서 피부 조직을 절개하고 7 톨루이딘 블루 염색에 의해 정량화 될 수있다. 비만 세포는 높은 친화도의 IgE 수용체 FcεRI를 발현 주요 지방 세포 집단 마찬가지로 비만 세포 탈과립은 종종 피부, IgE 매개 알레르기 반응을위한 마커로서 사용된다. 조직 염색에 넘쳐 분광 광도 측정을위한 기술은 1990 년 10 래트 및 마우스 (11)의 수동적 피부 아나필락시스 (PCA)를 위해 개발되었다. 다음 지역 아나필락시스 분석 프로토콜. 코지마 외에서 1을 적용하고, 알레르기 반응을 이끌어 내기위한 항원으로 닭 계란 난백 알부민 (OVA)를 사용 하였다. 그러나, 원한다면 이외 OVA 항원을 사용할 수있다. 분석은 다음 permeabilit 혈관의 변화를 모니터하는 에반스 블루 염료를 사용세포의 IgE 가교 및 히스타민의 방출을 비만으로 인해 발생하는 y를 입력합니다.

<table border="0" cellpadding="0" cellspacing="0" width="796">
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			<td height="21" style="height:21px;width:271px;">Name of Material/ Equipment</td>
			<td style="width:167px;">Company</td>
			<td style="width:135px;">Catalog Number</td>
			<td style="width:225px;">Comments/Description</td>
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			<td height="21" style="height:21px;width:271px;">BALB/c Mice</td>
			<td style="width:167px;">The Jackson Laboratory</td>
			<td style="width:135px;">651</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;width:271px;">PBS pH 7.4</td>
			<td style="width:167px;">Quality Biologic</td>
			<td style="width:135px;">114-058-101</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Ovalbumin</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:135px;">A5503-10G</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Imject Alum</td>
			<td style="width:167px;">Thermo Scientific</td>
			<td style="width:135px;">77161</td>
			<td style="width:225px;">Mix thoroughly before use</td>
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			<td height="21" style="height:21px;width:271px;">Evans Blue Dye</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:135px;">E2129</td>
			<td style="width:225px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Formamide</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:135px;">295876</td>
			<td style="width:225px;">99%+ Spectrophotometric grade</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Isoflurane, USP</td>
			<td style="width:167px;">Phoenix</td>
			<td style="width:135px;">NDC 57319-474-06</td>
			<td style="width:225px;"></td>
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			<td height="21" style="height:21px;width:271px;">1cc Insulin syringes</td>
			<td style="width:167px;">BD</td>
			<td style="width:135px;">329654</td>
			<td style="width:225px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:271px;">3/10 cc Insulin syringe with 31G needle</td>
			<td style="width:167px;">Terumo</td>
			<td style="width:135px;">NDC 100861</td>
			<td style="width:225px;"></td>
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			<td height="21" style="height:21px;width:271px;">27G Needles</td>
			<td style="width:167px;">BD</td>
			<td style="width:135px;">305109</td>
			<td style="width:225px;"></td>
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			<td height="21" style="height:21px;width:271px;">Forceps</td>
			<td style="width:167px;">F.S.T.</td>
			<td style="width:135px;">11000-12</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Surgical scissors</td>
			<td style="width:167px;">F.S.T.</td>
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			<td height="21" style="height:21px;width:271px;">5ml Polystyrene round-bottom tubes</td>
			<td style="width:167px;">BD Falcon</td>
			<td style="width:135px;">352058</td>
			<td style="width:225px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:271px;">1.5ml Microcentrifuge tubes</td>
			<td style="width:167px;">Medical Supply Partners</td>
			<td style="width:135px;">15-1151</td>
			<td style="width:225px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:271px;">15ml Conical tubes</td>
			<td style="width:167px;">BD Falcon</td>
			<td style="width:135px;">352097</td>
			<td style="width:225px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Flat-bottom 96 well plate</td>
			<td style="width:167px;">Costar</td>
			<td style="width:135px;">3590</td>
			<td style="width:225px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Scotch tape</td>
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			<td style="width:225px;"></td>
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			<td height="24" style="height:24px;width:271px;">RC2 Rodent Anesthesia System</td>
			<td style="width:167px;">VetEquip</td>
			<td style="width:135px;">922100</td>
			<td style="width:225px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Vortex Genie 2</td>
			<td style="width:167px;">Scientific Industries</td>
			<td style="width:135px;">SI-0236</td>
			<td style="width:225px;">Model G560 with 3 inch platform</td>
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measuring local anaphylaxis in mice

알레르기 반응은 비만 세포 및 호염기구, 및 혈관 작용 및 염증 매개체의 후속 릴리스의 활성화의 결과이다. 감작 된 개인의 알레르기 항원에 노출되면 약간의 홍반에서 생명을 위협하는 아나필락시스까지 다양 임상 증상이 발생할 수 있습니다. 실험실에서, 다양한 동물 모델은 알레르기 반응을 구동 메커니즘을 이해하기 위해 개발되었다. 여기서, 우리는 지역화 알레르기 반응을 정량화 혈관 투과성의 변화를 측정하기위한 자세한 방법을 설명합니다. 지역 아나필락시스 분석은 첫째 1920 년대에보고하고, 코지마 외 출판 기술에서 적응하고있다. 2007 년 한.이 분석에서, OVA로 감작 마우스는 차량 왼쪽 귀에 및 OVA와 오른쪽 귀에 도전한다. 이것은 에반스 블루 염료의 정맥 내 주사에 의해 이어진다. 텐 분 에반스 블루를 주입 한 후, 동물은 extravasated에있다 염료 안락사되고귀에 포름 아미드 하룻밤 추출된다. 추출 염료의 흡광도 후 분광 광도계 정량화된다. 이 방법은 확실하게 지역의 알레르기 반응의 시각 및 정량화 할 수있는 징후가 발생합니다.

성공적으로 분석을 시행 한 동물 피부와 푸른 눈을 표시해야합니다. PBS 감응 동물 따라서 양쪽 귀에 흰색 (그림 1A) 남아 있어야, PBS 또는 OVA 도전 중 하나에 반응하지 않아야합니다. OVA 감작 동물, PBS 챌린지를 수신하는 귀 (왼쪽)이 완전히 흰색 또는 주사 부위에서의 국소 방식으로 가볍게 푸른 여야한다. OVA 도전을 받고 귀 (오른쪽) 염료 관리 (그림 1B) 후 10 분 동안 점진?...

Watch this Scientific Journal Video about Measuring Local Anaphylaxis in Mice at JoVE.com

Measuring Local Anaphylaxis in Mice

비만 세포와 호염기구의 활성화를 특징으로 알레르기 반응은, IgE 항체의 가교 및 염증 매개 물질의 방출에 의해 구동된다. 알레르기 반응의 정량적 인 평가는 알레르겐 공격 후 혈관 투과성의 변화를 모니터하는 에반스 블루 염료를 사용함으로써 달성 될 수있다.

마우스에 현지 아나필락시스 측정

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

Research

JoVE Journal

Immunology and Infection

19.1K 조회수.  Uniformed Services University of the Health Sciences.  비만 세포와 호염기구의 활성화를 특징으로 알레르기 반응은, IgE 항체의 가교 및 염증 매개 물질의 방출에 의해 구동된다. 알레르기 반응의 정량적 인 평가는 알레르겐 공격 후 혈관 투과성의 변화를 모니터하는 에반스 블루 염료를 사용함으로써 달성 될 수있다. 

비디오: Measuring Local Anaphylaxis in Mice

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

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

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

유동세포계측법로 고정하고 살아있는 세포에서 Calpain 활동을 측정

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

연구

면역학 및 감염병학

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

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

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

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

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

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

Measurement of &#947;HV68 Infection in Mice

&gamma;-Herpesviruses (&gamma;-HVs) are notable for their ability to establish latent infections of lymphoid cells1. The narrow host range of human &gamma;-HVs, such as EBV and KSHV, has severely hindered detailed pathogenic studies. Murine &gamma;-herpesvirus 68 (&gamma;HV68) shares extensive genetic and biological similarities with human &gamma;-HVs and is a natural pathogen of murid rodents2. As such, evaluation of &gamma;HV68 infection of mice inbred strains at different stages of viral infection provides an important model for understanding viral lifecycle and pathogenesis during &gamma;-HVs infection.
Upon intranasal inoculation, &gamma;HV68 infection results in acute viremia in the lung that is later resolved into a latent infection of splenocytes and other cells, which may be reactivated throughout the life of the host3,4. In this protocol, we will describe how to use the plaque assay to assess infectious virus titer in the lung homogenates on Vero cell monolayers at the early stage (5 - 7 days) of post-intranasal infection (dpi). While acute infection is largely cleared 2 - 3 weeks postinfection, a latent infection of &gamma;HV68 is established around 14 dpi and maintained later on in the spleen of the mice. Latent infection usually affects a very small population of cells in the infected tissues, whereby the virus stays dormant and shuts off most of its gene expression. Latently-infected splenocytes spontaneously reactivate virus upon explanting into tissue culture, which can be recapitulated by an infectious center (IC) assay to determine the viral latent load. To further estimate the amount of viral genome copies in the acutely and/or latently infected tissues, quantitative real-time PCR (qPCR) is used for its maximal sensitivity and accuracy. The combined analyses of the results of qPCR and plaque assay, and/or IC assay will reveal the spatiotemporal profiles of viral replication and infectivity in vivo.

Measurement of &amp;#947;HV68 Infection in Mice

&#947;-Herpesviruses (&#947;-HVs) establish life-long persistency in their host. Infection of mice with &#947;-HV68 provides a genetically tractable in vivo model for the characterization of the lifecycle/pathogenesis of &#947;HVs. This protocol describes the detection and quantitation of &#947;HV68 infection at acute and latent stages following infection by plaque-forming, infectious center, and qPCR assays.

마우스에 γHV68 감염의 측정

&gamma;-Herpesviruses (&gamma;-HVs) are notable for their ability to establish latent infections of lymphoid cells1. The narrow host range of human ...

Intralymphatic Immunotherapy and Vaccination in Mice

Vaccines are typically injected subcutaneously or intramuscularly for stimulation of immune responses. The success of this requires efficient drainage of vaccine to lymph nodes where antigen presenting cells can interact with lymphocytes for generation of the wanted immune responses. The strength and the type of immune responses induced also depend on the density or frequency of interactions as well as the microenvironment, especially the content of cytokines. As only a minute fraction of peripherally injected vaccines reaches the lymph nodes, vaccinations of mice and humans were performed by direct injection of vaccine into inguinal lymph nodes, i.e.&#160;intralymphatic injection. In man, the procedure is guided by ultrasound. In mice, a small (5-10 mm) incision is made in the inguinal region of anesthetized animals, the lymph node is localized and immobilized with forceps, and a volume of 10-20 &#956;l of the vaccine is injected under visual control. The incision is closed with a single stitch using surgical sutures. Mice were vaccinated with plasmid DNA, RNA, peptide, protein, particles, and bacteria as well as adjuvants, and strong improvement of immune responses against all type of vaccines was observed. The intralymphatic method of vaccination is especially appropriate in situations where conventional vaccination produces insufficient immunity or where the amount of available vaccine is limited.

Prophylactic and therapeutic vaccination often fails to stimulate strong immune responses due to week drainage of the vaccine to lymph nodes and consequently poor involvement of immune cells. By direct injection of vaccine to lymph nodes, so-called intralymphatic injection, vaccine efficacy can be strongly improved and vaccine doses can be reduced.

마우스의 Intralymphatic 면역 및 예방 접종

Vaccines are typically injected subcutaneously or intramuscularly for stimulation of immune responses. The success of this requires efficient drainage of ...

Measuring Deformability and Red Cell Heterogeneity in Blood by Ektacytometry

Decreased red cell deformability is characteristic of several disorders. In some cases, the extent of defective deformability can predict severity of disease or occurrence of serious complications. Ektacytometry uses laser diffraction viscometry to measure the deformability of red blood cells subject to either increasing shear stress or an osmotic gradient at a constant value of applied shear stress. However, direct deformability measurements are difficult to interpret when measuring heterogenous blood that is characterized by the presence of both rigid and deformable red cells. This is due to the inability of rigid cells to properly align in response to shear stress and results in a distorted diffraction pattern marked by an exaggerated decrease in apparent deformability. Measurement of the degree of distortion provides an indicator of the heterogeneity of the erythrocytes in blood. In sickle cell anemia, this is correlated with the percentage of rigid cells, which reflects the hemoglobin concentration and hemoglobin composition of the erythrocytes. In addition to measuring deformability, osmotic gradient ektacytometry provides information about the osmotic fragility and hydration status of erythrocytes. These parameters also reflect the hemoglobin composition of red blood cells from sickle cell patients. Ektacytometry measures deformability in populations of red cells and does not, therefore, provide information on the deformability or mechanical properties of individual erythrocytes. Regardless, the goal of the techniques described herein is to provide a convenient and reliable method for measuring the deformability and cellular heterogeneity of blood. These techniques may be useful for monitoring temporal changes, as well as disease progression and response to therapeutic intervention in several disorders. Sickle cell anemia is one well-characterized example. Other potential disorders where measurements of red cell deformability and/or heterogeneity are of interest include blood storage, diabetes, Plasmodium infection, iron deficiency, and the hemolytic anemias due to membrane defects.

Here we present techniques to measure red cell deformability and cellular heterogeneity by ektacytometry. These techniques are applicable to general investigations of red cell deformability and specific investigations of blood diseases characterized by the presence of both rigid and deformable red cells in circulation, such as sickle cell anemia.

Deformability 및 Ektacytometry에 의해 혈액이 질 레드 셀 측정

Decreased red cell deformability is characteristic of several disorders. In some cases, the extent of defective deformability can predict severity 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 ...

Mouse Body Temperature Measurement Using Infrared Thermometer During Passive Systemic Anaphylaxis and Food Allergy Evaluation

Mouse body temperature measurement is of paramount importance for investigating allergies and anaphylactic symptoms. Rectal probes for temperature readings is common, and they have been proven to be accurate and invaluable in this regard. However, this method of temperature measurement requires the mice to be anesthetized in order to insert the probe without injury to the animal. This limits the ability to observe other phenotypes of the mouse simultaneously. In order to investigate other phenotypes while measuring temperatures, rectal probes are not ideal, and another method is desired. Here, we introduce a noninvasive method of temperature measurement that foregoes the requirement for mouse anesthesia while maintaining equal reliability to rectal probes in measuring body temperature. We use an infrared thermometer that detects body surface temperatures at ranges between 2 and 150 mm. This method of body temperature measurement is successful in reliably replicating temperature change trends during passive system anaphylaxis experiments in mice. We show that body surface temperatures are about 2.0 &#176;C lower than rectal probe measurements, but the degree of temperature drop follows the same trend. Furthermore, we use the same technique to observe mice in a food allergy model to evaluate temperature and activity levels simultaneously.

Here we present a new method to accurately measure body temperature differences in passive systemic anaphylaxis (PSA) and food allergy mouse models using an infrared thermometer. This procedure has been accurately duplicated in previous PSA results.

수동 체계 알레르기 및 식품 알레르기 평가 하는 동안 적외선 온도계를 사용 하 여 마우스 신체 온도 측정

Mouse body temperature measurement is of paramount importance for investigating allergies and anaphylactic symptoms. Rectal probes for temperature ...

Quantification of Atherosclerosis in Mice

Cardiovascular disease is the main cause of death in the world. The underlying cause in most cases is atherosclerosis, which is in part a chronic inflammatory disease. Experimental atherosclerosis studies have elucidated the role of cholesterol and inflammation in the disease process. This has led to successful clinical trials with pharmaceutical agents that reduce clinical manifestations of atherosclerosis. Careful and well-controlled experiments in mouse models of the disease could further elucidate the pathogenesis of the disease, which is not fully understood. Standardized lesion analysis is important to reduce experimental variability and increase reproducibility. Determining lesion size in aortic root, aortic arch, and brachiocephalic artery are common endpoints in experimental atherosclerosis. This protocol provides a technical description for evaluation of atherosclerosis at all these sites in a single mouse. The protocol is particularly useful when material is limited, as is frequently the case when genetically modified animals are being characterized.

Murine models of atherosclerosis are useful tools to investigate pathogenic pathways on a molecular level, but require standardized quantification of lesion development. This protocol describes an optimized method to determine lesion size in the major arterial vessels including the aortic root, aortic arch, and brachiocephalic artery.

마우스에서 동맥 경화증의 정량화

Cardiovascular disease is the main cause of death in the world. The underlying cause in most cases is atherosclerosis, which is in part a chronic ...

Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase (G6PI)-Induced RA Mice

Rheumatoid arthritis (RA) is a complex chronic inflammatory autoimmune disease. The pathogenesis of the disease is related to invariant natural killer T (iNKT) cells. Patients with active RA present fewer iNKT cells, defective cell function, and excessive polarization of Th1. In this study, an RA animal model was established using a mixture of hGPI325-339 and hGPI469-483 peptides. The iNKT cells were obtained by in vivo induction and in vitro purification, followed by infusion into RA mice for adoptive immunotherapy. The in vivo imaging system (IVIS) tracking revealed that iNKT cells were mainly distributed in the spleen and liver. On day 12 after cell therapy, the disease progression slowed down significantly, the clinical symptoms were alleviated, the abundance of iNKT cells in the thymus increased, the proportion of iNKT1 in the thymus decreased, and the levels of TNF-&#945;, IFN-&#947;, and IL-6 in the serum decreased. Adoptive immunotherapy of iNKT cells restored the balance of immune cells and corrected the excessive inflammation of the body.

Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase G6PI-Induced RA Mice

This protocol uses G6PI mixed peptides to construct rheumatoid arthritis models that are closer to that of human rheumatoid arthritis in CD4+ T cells and cytokines. High purity invariant natural killer T cells (mainly iNKT2) with specific phenotypes and functions were obtained by in vivo induction and in vitro purification for adoptive immunotherapy.

포도당-6-인산염 이사머라제(G6PI)-유도 RA 마우스에서 iNKT 세포의 입양 면역 요법

Rheumatoid arthritis (RA) is a complex chronic inflammatory autoimmune disease. The pathogenesis of the disease is related to invariant natural killer T ...

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are cleared from the human lung alveoli by being phagocytosed by innate monocytes and/or neutrophils. This protocol offers a fast and reliable measurement of phagocytosis by flow cytometry using fluorescein isothiocyanate (FITC)-labeled conidia for co-incubation with human leukocytes and subsequent counterstaining with an anti-FITC antibody to allow discrimination of internalized and cell-adherent conidia. Major advantages of this protocol are its rapidness, the possibility to combine the assay with cytometric analysis of other cell markers of interest, the simultaneous analysis of monocytes and neutrophils from a single sample and its applicability to other cell wall-bearing fungi or bacteria. Determination of percentages of phagocytosing leukocytes provides a means to microbiologists for evaluating virulence of a pathogen or for comparing pathogen wildtypes and mutants as well as to immunologists for investigating human leukocyte capabilities to combat pathogens.

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

This protocol provides a fast and reliable method to quantitatively measure phagocytosis of Aspergillus fumigatus conidia by human primary phagocytes using flow cytometry and to discriminate phagocytosis of conidia from mere adhesion to leukocytes.

유세포 측정을 사용하여 인간 백혈구에 의한 아스퍼질러스 훈증 코니디아의 식세포증 측정

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are ...