이 작품은 중국 국립 자연 과학 재단 (81573992)의 지원을 받았다. 그들의 언어 편집 지원에 대해 Emily K. Lo와 Kathleen DiNapoli에게 감사드립니다.

이 프로토콜은 상하이 한의대의 동물 관리 지침을 따르며 동물 실험 상하이 동물 윤리위원회의 승인을 받았습니다. 1. 프로토콜 I : EXD 준비 <ol><li> 계산 <ol><li> EXD를 6 주령의 ICR (female Imprinting Control Region) 마우스 (0.02 kg / mouse) 10 마리의 치료 그룹에 12 주 동안 주 5 회 투여하십시오. 참고 : 총 1.702 EXD가 필요합니다. EXD는 개별 증분으로 측정되기 때문에 실제로는 2 개의 EXD를 관리해야합니다. </li><li> 마우스 당 EXD 부피를 계산하십시오 ( 예 : V = (0.1418 EXD / kg • 일) * (0.02 kg / 마우스) * 50 mL / EXD = 0.14 mL / 마우스 • 일). </li></ol></li><li> 몸을 담그다 <ol><li> 두 가지 EXD ( 즉, Curculigo orchioides Gaertn (18g), Herba Epimedii (18g), Radix Morindae Officinalis (18g), RAdix Angelicae Sinensis (18g), Cortex Phellodendri (12g), Rhizoma Anemarrhenae (12g), 총 96g)를 뚜껑이 달린 용기에 넣습니다. 참고 : 세라믹 용기를 사용하는 것이 좋습니다. </li><li> 생 약초를 500 mL의 증류수로 1 시간 동안 담그고; 물은 허브를 약 1 인치까지 덮어야합니다. 모든 허브가 완전히 흡수되도록하십시오. </li></ol></li><li> 첫 번째 물 추출 <ol><li> 물이 끓을 때까지 (약 5 ~ 10 분, 시간은 가열 력, 용기 및 허브와 물의 양에 따라 달라질 때까지) 가스 밥솥이나 고주파 밥솥을 사용하여 약초를 가열하십시오. 2 시간 동안 낮은 끓는 물로 전원을 끕니다. </li></ol></li><li> 첫 번째 여과 <ol><li> 일반 여과지 또는 거즈와면을 사용하여 500mL 유리 비이커를 덮으십시오. 조심스럽게 필터를 통해 비누에 달걀을 붓습니다. 컨테이너에 허브를 남겨주세요. </li><li> 남은 허브 잔여 물을 반환하십시오.용기에 걸러진 여과지. </li></ol></li><li> 2 차 수분 섭취 <ol><li> 허브와 함께 증류수를 용기에 넣으십시오. 약 1 인치 정도의 약초를 가려야합니다. </li><li> 단계 1.3을 반복하십시오. </li></ol></li><li> 2 차 여과 <ol><li> 1.4.1 및 1.4.2 단계를 반복하십시오. </li><li> 같은 비이커에 두 번째 달인을 붓는다. 첫 번째와 두 번째 즙을 함께 섞는다. </li></ol></li><li> 집중 <ol><li> 비누를 사이에 석면이없는 철제 거즈가 달린 가스 밥솥에 올려 놓고 낮은 끓인 물로 가열하고 천천히 연속적으로 유리 막대로 저어 준다. </li><li> 달임이 200 mL로 줄어들면 500 mL 비이커로 옮긴다. </li><li> 달여가 100 mL가 될 때까지 1.7.1 단계를 반복하십시오. </li></ol></li><li> 보관 및 관리 <ol><li> 농축 된 달걀을 멸균 유리 병에 옮기십시오. 그것은 room temperatu로 식도록 내버려둬 라.re (RT). 장기간 보관할 경우 1 주일 이내에 -70 ° C에서 사용하는 경우 4 ° C에서 보관하십시오. </li><li> 위관 (gavage) 바늘을 사용하여 1 일 1 회, 12 주 동안 1 주일에 1 회, 난소 절제 (OVX) 마우스에 0.14 mL / 마우스 췌장을 투여하십시오. </li></ol></li></ol> 2. 프로토콜 II : EXD 함유 세럼의 제조 <ol><li> 계산 <ol><li> 각 쥐 ​​(1 개월)의 체중으로 0.15 kg을 사용하여 쥐 - 등가 선량 (RED)을 계산하십시오 : dB = (1/60) * (90/100) * (60 / 0.15) 1/3 = 0.111 EXD / kg / 일. 참고 : 100 mL의 배지 (10 % EXD 함유 혈청)를 준비하기 위해 총 10 mL의 EXD 함유 혈청이 필요합니다. 각 쥐는 2 mL의 혈청을 제공해야합니다. 따라서 하루 3 회 EXD 투여를 위해서는 6 마리의 쥐 (20 %의 손실을 감안)가 필요합니다. EXD의 수는 다음과 같습니다 : (0.111 EXD / kg • day) * (0.15 kg / rat) * (6 마리) * (3 일) = 0.300 EXD. 다시, EXD는 이산 증분으로 측정되기 때문에,1 EXD를 사용하십시오. </li><li> 쥐 ( 즉, V = (0.111 EXD / kg • day) * (0.15 kg / rat) * (50 mL / EXD) = 0.83 mL / rat • day) 당 적용된 EXD의 부피를 계산합니다. </li></ol></li><li> 몸을 담그다 <ol><li> 1 EXD 용 원료를 뚜껑이 달린 용기에 넣으십시오. 생약을 증류수에 1 시간 동안 담가 두십시오. 물은 허브를 약 1 인치까지 덮어야합니다. 모든 허브가 완전히 흡수되도록하십시오. </li></ol></li><li> 첫 번째 물 추출 <ol><li> 물이 끓을 때까지 고전력의 유도 밥솥을 사용하여 약초를 가열하십시오 (약 5 ~ 10 분, 시간은 화력, 용기 및 허브와 물의 양에 따라 다릅니다). 2 시간 동안 낮은 끓는 물로 전원을 끕니다. </li></ol></li><li> 첫 번째 여과 <ol><li> 일반 여과지 또는 거즈와면을 사용하여 500mL 유리 비이커를 덮으십시오. 조심스럽게 필터를 통해 비누에 달걀을 붓습니다. 컨테이너에 허브를 남겨주세요. </li><li&gt; 여과지에있는 생약 잔여 물을 용기에 넣으십시오. </li></ol></li><li> 2 차 수분 섭취 <ol><li> 허브와 함께 증류수를 용기에 넣으십시오. 약 1 인치 정도의 약초를 가려야합니다. </li><li> 2.4 단계를 반복하십시오. </li></ol></li><li> 2 차 여과 <ol><li> 2.5.1 및 2.5.2 단계를 반복하십시오. </li><li> 두 번째 달인을 2.4.1 단계에서와 같은 비이커에 붓고 첫 번째와 두 번째 데오스를 함께 섞는다. </li></ol></li><li> 집중 <ol><li> 비누를 사이에 석면없는 철사 거즈가 달린 가스 밥솥에 올려 놓고 낮은 끓인 물로 가열하고 유리 막대로 천천히 연속적으로 저어 준다. </li><li> 달이기가 100 mL가 될 때까지 100 mL 비이커에 옮긴다. </li><li> 달임이 50 mL가 될 때까지 2.7.1 단계를 반복하십시오. </li></ol></li><li> 보관 및 관리 <ol><li> 농축 된 달걀을 증기로 옮기십시오.ile 유리 병입니다. 그것을 RT로 식히자. 장기간 보관할 경우 1 주일 이내에 -70 ° C에서 사용하는 경우 4 ° C에서 보관하십시오. </li><li> 1 일 1 회, 0.83 mL / 래트를 3 일간 위내에 투여한다. </li></ol></li><li> EXD 함유 혈청 제제 <ol><li> EXD의 마지막 투여 후 1 시간에 300mg / 100g의 80mg / kg ketamine과 10mg / kg xylazine을 복강 내 주사하여 쥐를 마취시킨다. 발끝으로 적절한 마취를 확인하십시오. </li><li> 메스를 사용하여 쥐의 피부와 복막을 복부에서 흉부 바닥으로 절개하십시오 (또는 가위를 똑바로 세우십시오). 각각 약 5cm와 0.5cm의 길이와 깊이의 절개를 만듭니다. 티슈 페이퍼를 사용하여 복부 내장을 왼쪽으로 움직입니다. </li><li> 휴지를 사용하여 복부 대동맥의 결합 조직을 제거하여 혈관을 명확하게 노출시킵니다. </li><li> 10 ML, 22 게이지 주사기를 사용하여 복부 대동맥에서 천천히 혈액을 그리십시오. 전송 대상 :바늘을 제거한 후 15mL 멸균 튜브; 한 마리의 쥐가 8-10 mL의 혈액을 생산할 수 있습니다. 참고 : 쥐는 혈액이 채워지기 시작할 때 ( 즉, 복부 대동맥이 맥박이있는 상태에서) 살아 있어야합니다. </li><li> 방 RT에서 30-60 분 동안 똑바로 세워 혈액을 채 웁니다. 20 분 동안 500-600 XG에서 원심 분리기. 조심스럽게 모든 상등액 (혈청)을 50 mL 멸균 튜브 1 개에 넣고 여러 동물의 모든 혈청을 함께 섞으십시오. </li><li> 30 분 동안 56 ° C 수조에서 혈청을 배양하여 열 불 활성화를 수행하십시오. 0.22 μm의 공극 크기의 친수성 폴리 에테르 설폰 막을 가진 주사기 필터를 사용하여 혈청을 여과합니다. 신선한 혈청을 사용하거나 -20 ° C에서 보관하십시오. </li></ol></li><li> 대조군 약물 함유 혈청 제제 (대조군에서 사용) <ol><li> 1 일 1 회 3 일 동안 동일한 용량의 생리 식염수 (0.83 mL)로 각 랫트를 투여하십시오. 다른 단계는 2.9 단계와 동일합니다.</li></ol></li><li> 신청 <ol><li> EXD 함유 혈청 또는 약물 함유 혈청 10 mL를 각 100 mL의 배지에 첨가하고 1 % 페니실린 - 스트렙토 마이신 (PS) 23을 첨가합니다. </li></ol></li></ol>

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저자는 경쟁적인 금전적 이해 관계가 없다고 선언합니다.

최근 몇 년 동안 동부 지역의 임상 환경에서 수천 년 동안 적용 되어온 대체 의학의 한 유형 인 한약에 많은 관심이 쏟아졌습니다. 전통적인 약초 의학은 현대 의학의 &quot;벤치에서 침대로&quot;패턴과는 달리 처음에는 &quot;bed to bench&quot;패턴을 통해 자신의 메커니즘을 설명해야합니다. 이것은 벤치 단계에서 수행 된 검증과 새로 최적화 된 약물의 개발을위한 처리에 이어 수행 될 수 있습니다. 지?...

전통적인 약초의 연구와 응용에 대한 관심이 현재 커지고 있습니다. 화학 성분이 확실한 현대 의약품과는 달리, 허브 성분에는 미지 성분이 포함되어 있으며 활성 성분을 전달할 수 있도록 추출 과정이 필요합니다. 많은 연구가 잘 알려진 구조를 가진 하나의 작은 화합물을 전체 약초 또는 허브 공식의 대표자로 선택하려고 시도하지만 약리학 적 효능이나 메커니즘은 동등한 것으로 간주 될 수 없다. 핑거 프린팅 (fingerprinting)은 복잡한 허브 수식의 구성 성분을 분석 할 수 있지만, 일부 성분은 아직 명확하게 분석되지 않아 연구 3에서 모든 추출물을 결합 할 때 어려움을 겪습니다. 수많은 성분 / 추출물의 상호 작용은 약초의 치료 효과를 중재합니다. 이러한 장점을 유지하기 위해 전통적인 추출물 형태이온은 여전히 ​​클리닉에서 널리 사용됩니다. 추출 과정은 치료 효능에 큰 영향을 미치기 때문에, 특히 생체 내 연구 4,5 에서 전통적 물 추출법의 표준 프로토콜이 필요합니다. 반면에, 약리학 적 기작을 탐색 할 때, 생체 외 또는 생체 외 연구 중에 약초를 처치하는 것은 도전 과제입니다. 약물 함유 혈청의 개념은 1988 년 Tashino에 의해 처음 제안되었다. 그 이후로 점점 많은 연구자들이 한약 7 , 8 , 9 에 그것을 적용 해왔다. 약물 함유 혈청의 방법은 혈청의 특정 성분의 영향과 같은 몇 가지 제한이 있지만, 여전히 생리 학적 조건을 밀접하게 모방하는 방법으로 간주됩니다. 10 , 11 , 12 , 13 을 완화하기 위해 1950 년대 초에 개발되었습니다. 또한 재생 불량성 빈혈 14 , 폐경기 골다공증 15 , 16 , 17 , 조기 난소 부전 18 , 유방암 19 , 난소 암 20 및 사춘기 지연 21에도 적용 되었습니다. 여기, 우리는 에르 - 시안 (erxian) 추출 (EXD)과 그 약물 함유 혈청의 제조를위한 상세한 프로토콜을 제시합니다. 또한 폐경기 골다공증 모델에 EXD 및 EXD를 함유 한 혈청을 적용하는 방법을 설명합니다. EXD는 Curculigo orchioides Gaertn , Herba Epimedii </eRadix Morindae Officinalis , Radix Angelicae Sinensis , Cortex Phellodendri 및 Rhizoma Anemarrhenae 각각 6 g을 하루에 성인 1 인당 투여한다. 마우스의 등가 선량은 다음 식 22 에 따라 0.1418 EXD / kg / day입니다. dB = dA * RB / RA * (WA / WB) 1/3 . dA와 dB는 각각 사람과 마우스의 체중 당 투여 량 (mg / kg)을 의미합니다. 용량 (mg / kg)은 EXD / kg의 수로 대체됩니다. RA와 RB는 각각 신체 표면적 (m 2 ) / 체중 (kg) 2/3 ( 표 1 참조)에 비례하는 인간 신체 계수와 마우스 몸 계수를 나타냅니다. WA 및 WB는 각각 사람 ​​및 마우스의 체중 (kg)을 나타낸다.

<table border="0" cellpadding="0" cellspacing="0" width="651">
	
	<tbody>
		<tr height="165">
			<td height="165" style="height:165px;width:216px;">Curculigo orchioides Gaertn (9&#8201;g), Herbaa Epimedii (9&#8201;g), Radix Morindae Officinalis (9&#8201;g), Radix Angelicae Sinensis (9&#8201;g), Cortex Phellodendri (6&#8201;g), and Rhizoma Anemarrhenae (6&#8201;g)</td>
			<td style="width:149px;">Kang-qiao Chinese Medicine Yinpian Co. Ltd (Shanghai, CN)</td>
			<td style="width:119px;">160922</td>
			<td style="width:167px;">EXD components</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Filter paper</td>
			<td style="width:149px;">GElifesciences</td>
			<td style="width:119px;">99-103-952</td>
			<td style="width:167px;">Filter EXD decoction before concentration</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:216px;">Imprinting Control Region (ICR) mice</td>
			<td style="width:149px;">Shanghai Laboratory Animal Center</td>
			<td style="width:119px;">SCXK 2007-0005</td>
			<td style="width:167px;">In vivo study</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:216px;">Sprague Dawley rats</td>
			<td style="width:149px;">Shanghai Laboratory Animal Center</td>
			<td style="width:119px;">SCXK 2007-0005</td>
			<td style="width:167px;">EXD-containing serum preparation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Syringe filter</td>
			<td style="width:149px;">Millipore</td>
			<td style="width:119px;">SLGP033RB</td>
			<td style="width:167px;">0.22 &#181;m</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">gavage needles (10 ml)</td>
			<td style="width:149px;">Shanghai BO Ge trade sales department</td>
			<td align="right" style="width:119px;">59104274</td>
			<td style="width:167px;">Adminstration of EXD</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">Ketamine (80 mg/kg)&#160;</td>
			<td style="width:149px;">Fujian Gutian Pharma Co. Ltd</td>
			<td style="width:119px;">H35020148</td>
			<td>Anesthesia</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Xylazine (10 mg/kg)</td>
			<td style="width:149px;">Sunway Pharma Co. Ltd</td>
			<td style="width:119px;">CB07591</td>
			<td>Anesthesia</td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:216px;">Dulbecco&#8217;s modification of Eagle&#8217;s medium Dulbecco (DMEM) culture medium</td>
			<td style="width:149px;">Gibco</td>
			<td style="width:119px;">12800-116</td>
			<td style="width:167px;">DMEM with 2 mM L-glutamine and without ribonucleosides and ribonucleotides</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Streptomycin</td>
			<td style="width:149px;">Sigma</td>
			<td align="right" style="width:119px;">1277</td>
			<td style="width:167px;">100 &#181;g / ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Penicillin</td>
			<td style="width:149px;">Sigma</td>
			<td align="right" style="width:119px;">4687</td>
			<td style="width:167px;">100 &#181;g / ml</td>
		</tr>
	</tbody>
</table>

preparation of herbal medicine: er-xian decoction and er-xian-containing serum for in vivo and in vitro experiments

임상 약제로 사용되는 대체 의학 인 한약은 최근 몇 년 동안 주목을 받아 왔습니다. 인체에 전달하기 전에 원료 약초에서 활성 성분을 방출하기 위해 일반적으로 추가 추출 절차가 필요합니다. 수분 섭취는 여전히 임상 환경에서 광범위하게 사용되는 고전적인 추출 과정입니다. 여기에서는 실험적 연구에 약초 즙을 적용하기 위해 erxian decoction (EXD)에 대한 상세한 프로토콜을 제안합니다. EXD의 4 가지 주요 단계 (수적, 수분 섭취, 여과 및 농축)뿐만 아니라 동물에 적합한 용량의 계산이 기술되어있다. 또한, 혈청 함유 EXD는 생체 외 검증을위한 수단으로 쥐에게 도입됩니다. 여기에 쥐에게 EXD를 3 일간 경구 투여 하였다. 혈액 샘플을 채취하고, 불 활성화시키고, 원심 분리하고, 여과시켰다. 배양 배지로 희석 된 혈청은 세포 또는 조직 을 vitro. 예를 들어, EXD는 in vivo 및 in vitro 연구에 모두 적용되었으며 EXD가 골 형성을 향상시키는 것으로 나타났습니다. 이 프로토콜은 약초의 제조 및 응용에 대한 참고 자료로 사용할 수 있습니다.

EXX가 OVX 마우스의 골밀도에 미치는 영향 요추 척수 절편의 Hematoxylin과 eosin (H &amp; E) 염색은 OVX 군 ( 그림 1A , 왼쪽 패널)과 비교하여 생체 내 EXD 치료 후 증가 된 골소주 ( 그림 1A , 오른쪽 패널)를 보여줍니다. 그림 1B 는 OVX 마우스 ( 그림 1B , 왼쪽 패널)와 EXX 치료 12 주 ( <st...

Watch this Scientific Journal Video about Preparation of Herbal Medicine: Er-Xian Decoction and Er-Xian-containing Serum for In Vivo and In Vitro Experiments at JoVE.com

Preparation of Herbal Medicine: Er-Xian Decoction and Er-Xian-containing Serum for In Vivo and In Vitro Experiments

여기에서는 4 가지 단계 (담금질, 달인, 여과 및 농축)의 erxian decoction (EXD) 준비 과정을 제시하고 준비된 EXD 함유 혈청을 쥐에게 투여하는 방법을 보여줍니다. 이 방법은 생약과 생체 외 연구에 적용 할 수 있습니다.

약초의 준비 : Er-Xian의 달임과 Er-Xian 함유 세럼<em&gt; In Vivo</em&gt; 및<em&gt; 체외에서</em&gt; 실험

Traditional herbal medicine, an alternative medicine in the clinical setting, has received increased attention in recent years. Before delivery to the ...

preparation-herbal-medicine-er-xian-decoction-er-xian-containing

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JoVE Journal

Medicine

Preparation of Herbal Medicine: Er-Xian Decoction and Er-Xian-containing Serum for In Vivo and In Vitro Experiments

10.9K 조회수.  Shanghai University of Traditional Chinese Medicine.  여기에서는 4 가지 단계 (담금질, 달인, 여과 및 농축)의 erxian decoction (EXD) 준비 과정을 제시하고 준비된 EXD 함유 혈청을 쥐에게 투여하는 방법을 보여줍니다. 이 방법은 생약과 생체 외 연구에 적용 할 수 있습니다. 

비디오: Preparation of Herbal Medicine: Er-Xian Decoction and Er-Xian-containing Serum for In Vivo and In Vitro Experiments

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

세포 추적을위한 생체 19 F MRI에서

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

연구

의학

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

Due to the high level of heterogeneity and mutations inherent in human cancers, single agent therapies, or combination regimens which target the same pathway, are likely to fail. Emphasis must be placed upon the inhibition of pathways that are responsible for intrinsic and/or adaptive resistance to therapy. An active field of investigation is the development and testing of DNA repair inhibitors that promote the action of, and prevent resistance to, commonly used chemotherapy and radiotherapy. We used a novel protocol to evaluate the effectiveness of BRCA2 inhibition as a means to sensitize tumor cells to the DNA damaging drug cisplatin. Tumor cell metabolism (acidification and respiration) was monitored in real-time for a period of 72 hr to delineate treatment effectiveness on a minute by minute basis. In combination, we performed an assessment of metastatic frequency using a chicken embryo chorioallantoic membrane (CAM) model of extravasation and invasion. This protocol addresses some of the weaknesses of commonly used in vitro and in vivo methods to evaluate novel cancer therapy regimens. It can be used in addition to common methods such as cell proliferation assays, cell death assays, and in vivo murine xenograft studies, to more closely discriminate amongst candidate targets and agents, and select only the most promising candidates for further development.

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

Here, real-time monitoring of tumor cell metabolism, combined with an in vivo chicken embryo chorio-allantoic membrane (CAM) model of metastasis, are used to evaluate novel anti-cancer targets/agents for their ability to sensitize tumor cells to DNA damaging chemotherapeutics.

암 약물 과민 반응의 효과를 평가<em&gt; 체외</em&gt; 및<em&gt; 생체</em

Due to the high level of heterogeneity and mutations inherent in human cancers, single agent therapies, or combination regimens which target the same ...

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence (NIRF) xenograft imaging experiments in mice using fluorophore labelled nanobodies and conventional antibodies.
First we describe how to generate subcutaneous tumors in mice, using antigen-negative cell lines as negative controls and antigen-positive cells as positive controls in the same mice for intraindividual comparison. We outline how to administer intravenously near-infrared fluorophore labelled (AlexaFluor680) antigen-specific nanobodies and conventional antibodies. In vivo imaging was performed with a small-animal NIRF-Imaging system. After the in vivo imaging experiments the mice were sacrificed. We then describe how to prepare the tumors for parallel ex vivo analyses by flow cytometry and fluorescence microscopy to validate in vivo imaging results.
The use of the near-infrared fluorophore labelled nanobodies allows for non-invasive same day imaging in vivo. Our protocols describe the ex vivo quantification of the specific labeling efficiency of tumor cells by flow cytometry and analysis of the distribution of the antibody constructs within the tumors by fluorescence microscopy. Using near-infrared fluorophore labelled probes allows for non-invasive, economical in vivo imaging with the unique ability to exploit the same probe without further secondary labelling for ex vivo validation experiments using flow cytometry and fluorescence microscopy.

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence xenograft imaging experiments in mice using fluorophore labelled nanobodies and conventional antibodies.

나노 바디 및 항체 기반의 검증<em&gt; 생체</em마우스에서&gt; 종양 이종 이식 NIRF 이미징 실험 사용<em&gt; 전의 VIVO</em&gt; 유동 세포 계측법 및 현미경

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence (NIRF) xenograft imaging experiments in mice ...

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. Despite the discovery of key regulators of uterine contractility, this process is still not fully understood. Transgenic mice provide an ideal model in which to study parturition. Previously, the only method to study uterine contractility in the mouse was ex vivo isometric tension recordings, which are suboptimal for several reasons. The uterus must be removed from its physiological environment, a limited time course of investigation is possible, and the mice must be sacrificed. The recent development of radiometric telemetry has allowed for longitudinal, real-time measurements of in vivo intrauterine pressure in mice. Here, the implantation of an intrauterine telemeter to measure pressure changes in the mouse uterus from mid-pregnancy until delivery is described. By comparing differences in pressures between wild type and transgenic mice, the physiological impact of a gene of interest can be elucidated. This technique should expedite the development of therapeutics used to treat myometrial disorders during pregnancy, including preterm labor.

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

This manuscript provides a protocol for implanting telemeters in the mouse for the purpose of measuring intrauterine pressures during pregnancy.

자궁 원격 측정은 마우스 수축 압력을 측정하는<em&gt; 생체</em

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

인간 종양 이종 이식에서 마우스 세포의 고갈은 크게 대상 세포의 다운 스트림 분석을 향상

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic study in the liver. Here, numerous assays are described to comprehensively measure the phenotype and parameters of hepatic steatosis in mouse and hepatocyte models. 
Combining the physiological, histological, and biochemical methods, this system can be used to assess the progress of hepatic steatosis. In vivo, the measurements of body weight and nuclear magnetic resonance (NMR) provide a general understanding of mice in a non-invasive manner. Hematoxylin and Eosin (H&amp;E) and Oil Red O staining determine the histological morphology and lipid deposition of liver tissue under nutrient overload conditions, such as high-fat diet feeding. Next, the total lipid contents are isolated by chloroform/methanol extraction, which are followed by a biochemical analysis for triglyceride and cholesterol. Moreover, mouse primary hepatocytes are treated with high glucose plus insulin to stimulate lipid accumulation, an efficient in vitro model to mimic diet-induced hyperglycemia and hyperinsulinemia in vivo. Then, the lipid deposition is measured by Oil Red O staining and chloroform/methanol extraction. Oil Red O staining determines intuitive hepatic steatotic phenotypes, while the lipid extraction analysis determines the parameters that can be analyzed statistically. The present protocols are of interest to scientists in the fields of fatty liver diseases, insulin resistance, and type 2 diabetes.

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Here, optimized methods to generate in vivo and in vitro models of hepatic steatosis and to analyze the steatotic phenotypes and related physiological parameters are described.

의 최적화 분석<em&gt; 생체</em&gt; 및<em&gt; 체외</em&gt; 간 지방증

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic ...

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene and cell therapies are highly promising alternative approaches for CVD treatment. However, the broad clinical application of gene therapy is greatly limited by the lack of suitable gene delivery systems. The development of appropriate gene delivery vectors can provide a solution to current challenges in cell therapy. In particular, existing drawbacks, such as limited efficiency and low cell retention in the injured organ, could be overcome by appropriate cell engineering (i.e., genetic) prior to transplantation. The presented protocol describes the efficient and safe transient modification of endothelial cells using a polyethyleneimine superparamagnetic magnetic nanoparticle (PEI/MNP)-based delivery vector. Also, the algorithm and methods for cell characterization are defined. The successful intracellular delivery of microRNA (miR) into human umbilical vein endothelial cells (HUVECs) has been achieved without affecting cell viability, functionality, or intercellular communication. Moreover, this approach was proven to cause a strong functional effect in introduced exogenous miR. Importantly, the application of this MNP-based vector ensures cell magnetization, with accompanying possibilities of magnetic targeting and non-invasive MRI tracing. This may provide a basis for magnetically guided, genetically engineered cell therapeutics that can be monitored non-invasively with MRI.

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

This manuscript describes the efficient, non-viral delivery of miR to endothelial cells by a PEI/MNP vector and their magnetization. Thus, in addition to genetic modification, this approach allows for magnetic cell guidance and MRI detectability. The technique can be used to improve the characteristics of therapeutic cell products.

준비 및<em&gt; 체외</em&gt; 자화 특성의 내피 세포를 미르 변성

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene ...

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a wide variety of disorders including metabolic diseases, premature aging syndromes, and neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Maintenance of mitochondrial health depends on mitochondrial biogenesis and the efficient clearance of dysfunctional mitochondria through mitophagy. Experimental methods to accurately detect autophagy/mitophagy, especially in animal models, have been challenging to develop. Recent progress towards the understanding of the molecular mechanisms of mitophagy has enabled the development of novel mitophagy detection techniques. Here, we introduce several versatile techniques to monitor mitophagy in human cells, Caenorhabditis elegans (e.g., Rosella and DCT-1/ LGG-1 strains), and mice (mt-Keima). A combination of these mitophagy detection techniques, including cross-species evaluation, will improve the accuracy of mitophagy measurements and lead to a better understanding of the role of mitophagy in health and disease.

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitophagy, the process of clearing damaged mitochondria, is necessary for mitochondrial homeostasis and health maintenance. This article presents some of the latest mitophagy detection methods in human cells, Caenorhabditis elegans, and mice.

생체 외에서 그리고 Vivo에서 인간 세포, C. 선 충, 쥐에서 Mitophagy의 검출

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a ...

Preparation Of Gushukang (GSK) Granules for In Vivo and In Vitro Experiments

Traditional Chinese herbal medicine plays a role as an alternative method in treating many diseases, such as postmenopausal osteoporosis (POP). Gushukang (GSK) granules, a marketed prescription in China, have bone-protective effects in treating POP. Before administration to the body, one standard preparation procedure is commonly required, which aims to promote the release of active constituents from raw herbs and enhance the pharmacological effects as well as therapeutic outcomes. This study proposes a detailed protocol for using GSK granules in in vivo and in vitro experimental assays. The authors first provide a detailed protocol to calculate the animal-appropriate dosages of granules for in vivo investigation: weighing, dissolving, storage, and administration. Second, this article describes protocols for micro-CT scanning and the measurement of bone parameters. Sample preparation, protocols for running the micro-CT machine and quantification of bone parameters were evaluated. Third, serum-containing GSK granules are prepared, and drug-containing serum is extracted for in vitro osteoclastogenesis and osteoblastogenesis. GSK granules were intragastrically administered twice per day to rats for three consecutive days. Blood was then collected, centrifuged, inactivated, and filtered. Finally, serum was diluted and used for performing osteoclastogenesis and osteoblastogenesis. The protocol described here can be considered a reference for pharmacological investigations of herbal prescription medicines, such as granules.

Preparation Of Gushukang GSK Granules for In Vivo and In Vitro Experiments

This article provides a detailed protocol for preparing a working solution of Gushukang granules for animal studies and GSK granule containing serum for in vitro experiments. This protocol can be applied to pharmacological investigations of herbal medicines as well as prescriptions for both in vivo and in vitro experiments.

구슈강(GSK) 생체내 및 체외 실험을 위한 과립 준비

Traditional Chinese herbal medicine plays a role as an alternative method in treating many diseases, such as postmenopausal osteoporosis (POP). Gushukang ...

약초의 준비 : Er-Xian의 달임과 Er-Xian 함유 세럼

요약

더 많은 비디오 탐색

이 비디오의 챕터

세포 추적을위한 생체 ¹⁹ F MRI에서

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

암 약물 과민 반응의 효과를 평가

나노 바디 및 항체 기반의 검증

자궁 원격 측정은 마우스 수축 압력을 측정하는

인간 종양 이종 이식에서 마우스 세포의 고갈은 크게 대상 세포의 다운 스트림 분석을 향상

의 최적화 분석

준비 및

생체 외에서 그리고 Vivo에서 인간 세포, C. 선 충, 쥐에서 Mitophagy의 검출

구슈강(GSK) 생체내 및 체외 실험을 위한 과립 준비