우리는 애니 B. 크리스텐센, 엘리자베스 V. 안데르센, 벨린다 B. Bringtoft 및 실험 의학, 프레데릭 스 버그 캠퍼스, 보건 의료 학부, 덴마크 코펜하겐 대학의학과에서 동물 관리인의 숙련 된 지원을 인정합니다. 야곱 Lønholdt는 절차를 촬영에서 자신의 뛰어난 기술 지원에 대해 감사한다. 이 연구는 생체 내 약리학과 코펜하겐의 대학에 대한 LIFEPHARM 센터에 의해 부분적으로 지원되었다.

표시된 절차는 식품, 농업 및 수산부에서 덴마크의 동물 실험 검사관에 의해 승인 프로토콜에 따라 수행 하였다. 측면 복재 또는 눈꺼풀 정맥에서 1 혈액 샘플링 <ol><li> 마취없이이 절차와 하나의 조사, 기니피그를 처리 하나, 그리고 하나의 샘플을 수집의 도움을 수행합니다. </li><li> 혈관이 표시 될 때까지 전기 면도기와 뒷다리의 눈꺼풀 영역을 면도. 영역 천자 사이트 방해 모피 않도록 충분히 크다 확인. </li><li> 미지근한 물을 욕조에 뒷다리를 놓고 조심스럽게 혈관이 더 팽창하고 분명하게 다리를 문질러. </li><li> 깨끗한 수건으로 다리를 닦은 거즈 또는 70 % 알코올로 적신 면봉으로 구멍 사이트를 소독. </li><li> 기니피그을 억제하고 즉시 무릎 관절 t 위에 부드럽게 가압하면서 하방으로 연장 뒷다리오 정체를 제공합니다. </li><li> 21 G 바늘이나 란셋을 사용하여 정맥에 구멍. 혈액 등을 수집합니다., 수집 또는 모세관 튜브를 사용하여. 조심스럽게 혈액 흐름을 돕기 위해 다리를 조작. </li><li> 샘플이 획득 된 후, 정체를 놓습니다. 출혈이 멈출 때까지 천자 사이트에 부드러운 압력을 거즈 나 탈지면의 조각을 적용합니다. </li><li> 지혈을 보장하기 위해 5 ~ 10 분 동안 케이지와 모니터에 동물을 반환합니다. </li></ol> 귀 정맥에서 2 혈액 샘플링 <ol><li> 한 동료의 도움으로 단독으로 또는 바람직 마취없이이 절차를 수행합니다. </li><li> 거즈 또는 70 % 알코올에 적신 면봉을 사용하여 귀에의 등 표면을 소독. </li><li> 정체를 제공하는 귀베이스에 가벼운 압력을 적용합니다. </li><li> 21 G 바늘이나 란셋을 사용하여 정맥에 구멍을하고, 혈액을 수집. </li><li> , 정체를 해제 샘플을 획득하고 나면, GE와 거즈 나 탈지면의 조각을 적용출혈까지 천자 사이트에 ntle 압력이 중지되었습니다. </li><li> 지혈을 보장하기 위해 5 ~ 10 분 동안 케이지와 모니터에 동물을 반환합니다. </li></ol> 경정맥에서 3 혈액 샘플링 <ol><li> 하나의 추가 조사의 도움을 사용하여이 절차를 수행; 하나 기니피그 처리 한 샘플을 수집. </li><li> 이 절차의 전체 기간 동안 적합한 마취제로 기니 피그를 마취시키다. tiletamine의 혼합물 (0.93 ㎎ / ㎏), zolazepam (0.93 ㎎ / ㎏), 자일 라진 (1.49 ㎎ / ㎏) 및 butorphanol (0.06 ㎎ / ㎏)을 사용할 수 있습니다. </li><li> 그 뒷면에있는 마취 기니 피그를 놓고 조심스럽게 꼬리 쪽 앞다리를 철회. </li><li> 면도하고 소독 (예., 70 % 알코올로) 목의 복부 부분. </li><li> 코트 응고 위험을 감소시키기 위해 헤파린 또는 EDTA 샘플링 이전에 하나와 주사기. </li><li> 바늘 하나 ML의 주사기 AP 통신에 장착 (23 G는 나) 쇄골을 만져와 25 G를 삽입에 근접하여 1cm는 어깨 관절의 수준 정중선 측방. 약간의 부정적인 압력을가하면서 약간 위쪽과 중간 각도에서 주사기를 유지합니다. 경정맥이 매우 피상적​​ 위치해로 기본 구조물을 훼손하지 마십시오. 정맥의 증가 정체를 제공하기 위해 약간 뒤로 기니 돼지의 목을 기울입니다. </li><li> 혈액 샘플이 얻어진다 앞다리를 해제하고, 바늘이 후퇴 전에 정체를 경감. 천자 사이트에 부드러운 압력을 적용 거즈 나 탈지면의 건조 조각을 사용합니다. </li><li> 복구 케이지에 동물을 반환하고 마취에서 지혈 및 복구를 보장하기 위해 모니터링 할 수 있습니다. </li></ol>

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저자가 공개하는 게 없다. 모든 절차는 유럽 협약 ETS (123)의 이사회에 따른다 덴마크의 동물 실험 법에 의해 승인되었습니다.

마우스 및 래트에 비해, 기니피그 자연에 의해 공포되고 침착 접근 및 스트레스를 줄이기 위해 올바르게 처리되어야한다. 보기의 동물 복지의 관점에서,주의 행동을 자연스럽게 우선 순위; 그러나,이도. 수집 된 데이터, 예를 들면에 불안 - 관련 효과의 위험을 줄일 스테로이드 증가한 혈중 농도, 12 글루코스 것이다. 마찬가지로, 일일 처리는 정상적인 절차에 동물을 습관화하고 더?...

기니 피그 인해 비타민 C, 인간의 태반과 함께 효소와 지질 단백질 프로파일뿐만 아니라 공유 유사성을 대사 비교 혈장 단백의 영양 공급을위한 요구 사항으로 인체에 생물학적 유사성의 숫자로 가치 및 검증 실험 동물 모델 태아 개발 1-3,7,8. 이 기니피그 등의 비 알코올 지방간 질환, 심혈관 질환과 비타민 C 결핍증의 추정 효과뿐만 아니라, 예를 들면의 효과로 다이어트 관련 질병의 효과 연구를위한 매력적이고 적합한 모델을 만든다. 산모식이 개입을 개발 자손. 반복 채혈은 종종 생체 내 연구에서 모두 장기 및 단기 필요합니다. 인해 종 특이 해부학 차이 샘플링 기술의 변조는 다른 설치류 모델에 비해 기니피그의 혈액 시료를 얻기 위해 필요 <sup&gt; 4,5. 따라서 샘플링 및 처리 모두에 대한 상이한 요건 및 옵션을 가지고, 이는 정맥과 동맥의 과정에서 변화를 포함하고, 또한 그 기니아 피그 테일이 없다. 복재 또는 눈꺼풀 정맥에서 샘플링, 동물에 최소한의 불편과 스트레스를 유발하고 3,9 마취가 필요하지 않습니다. 이 기술은 혈액 (100 ~ 400 μL), 예를 들면 소량의 샘플을 반복 할 수 있습니다., 혈장 생화학 적 마커를 결정합니다. 더 작은 샘플 크기 (50 내지 100 μL)에 충분한 경우,이 아주 쉽게 귀 정맥을 천공하여 얻을 수있다. 이 기술은 또한 의식적 동물에서 수행하고 10,11 혈당을 측정 할 때, 예를 들면 유용 할 수있다. 경정맥에서 샘플링은 혈액의 많은 양 (1 ~ 2 ㎖)의 수집을 위해 수 있지만 마취가 필요합니다. 여기에, 우리는 의식이나 마취 기니 돼지 중 하나에 대 한 4 개의 서로 다른 혈액 샘플링 기법을 제시한다. PROCedures 않은 모든 터미널 절차는 실험 동물 (6) 혈액 수집을위한 가이드 라인을 초과하지 않는 양의 샘플 및 샘플의 수를 제공합니다. 그것은 스트레스를 최소로 유지해야한다는 동물 복지뿐만 아니라 좋은 과학의 이해 관계에 있습니다. 따라서, 단지 잘 훈련 된 유능한 개인은 동물의 절차를 수행해야합니다.

<table border="0" cellpadding="0" cellspacing="0" width="944">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:295px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:315px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Electric shaver</td>
			<td style="width:295px;">Wella (Contura), Denmark</td>
			<td style="width:119px;">273207</td>
			<td>Alternative product may be used. Multiple suppliers</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Syringes&#160;</td>
			<td style="width:295px;">B.Braun-Omnifix-F, Germany</td>
			<td style="width:119px;">9161406V</td>
			<td>Alternative product may be used. Multiple suppliers</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Needles</td>
			<td style="width:295px;">BD Microlance,Spain</td>
			<td style="width:119px;">301156</td>
			<td>Alternative product may be used. Multiple suppliers</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Microvette collection tube&#160;</td>
			<td style="width:295px;">Sarstedt, D-51588N&#252;mbrecht, Germany</td>
			<td style="width:119px;">201,288,100</td>
			<td>Alternative product may be used. Multiple suppliers</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Accu-Check analyzer</td>
			<td style="width:295px;">Roche A/S Diagnostics, Denmark</td>
			<td style="width:119px;"></td>
			<td>Alternative product may be used. Multiple suppliers</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Heparin 5000 IE/ml</td>
			<td style="width:295px;">LEO Pharma, Denmark</td>
			<td style="width:119px;"></td>
			<td>Alternative product may be used. Multiple suppliers</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Anesthesia</td>
			<td style="width:295px;">Zoletil 50, Chemvet, Denmark</td>
			<td style="width:119px;"></td>
			<td>Alternative product may be used. Multiple suppliers</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Anesthesia</td>
			<td style="width:295px;">Narcoxyl vet 20 mg/ml, Intervet, Denmark</td>
			<td style="width:119px;"></td>
			<td>Alternative product may be used. Multiple suppliers</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Anesthesia</td>
			<td style="width:295px;">Torbugesic vet 10 mg/ml, Scanvet, Denmark</td>
			<td style="width:119px;"></td>
			<td>Alternative product may be used. Multiple suppliers</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Neutral eye ointment</td>
			<td style="width:295px;"></td>
			<td style="width:119px;"></td>
			<td>Alternative product may be used. Multiple suppliers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gauze or a cotton swab&#160;</td>
			<td style="width:295px;"></td>
			<td style="width:119px;"></td>
			<td>Alternative product may be used. Multiple suppliers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Disinfectant (70% alcohol)</td>
			<td style="width:295px;"></td>
			<td style="width:119px;"></td>
			<td>Alternative product may be used. Multiple suppliers</td>
		</tr>
	</tbody>
</table>

non-terminal blood sampling techniques in guinea pigs

기니 돼지는 인간에게 여러 가지 생물학적 유사성을 가지고 실험 동물 모델 1-3을 검증하고 있습니다. 특정 방법론 대응 부족에 그러나, 기니아 피그의 사용은 현재 연구와 설명 데이터의 비교적 좁은 영역을 나타냅니다. 기니피그의 해부학 적 특징은 마우스 및 래트에 비해. 따라서 샘플링 기법을 조정함으로써 예컨대, 종 특이 차이를 수용 할 수 있도록, 다른 설치류 모델과 다소 상이 충분히 고품질의 샘플들을 획득하기 위해 필요하다. 생체 내 연구에서 모두 장기 및 단기 자주 샘플링 혈액을 반복하여 필요한만큼 기술의 선택은 물론 동물에서 응력 및 불편 함을 감소시키기 위해 고려해야 할뿐만 아니라 시료의 크기 및 접근의 요구 생존뿐만 아니라 준수하도록. 정맥혈 샘플 기니피그 등의 다수 위치에서 얻을 수있다., 복재 및 경정맥을정맥, 장점과 단점을 모두 포함하는 각 기술 4,5. 여기에, 우리는 의식이나 마취 기니 돼지 중 하나에 대 한 4 개의 서로 다른 혈액 샘플링 기법을 제시한다. 절차는 모든 비 터미널 절차는 실험 동물 (6) 혈액 수집을위한 가이드 라인을 초과하지 않는 양의 샘플 및 샘플의 수를 제공합니다. 모든 설명 방법은 철저한 테스트를 거쳤으며 우리의 연구 시설에서 연구에서 생체 내 혈액 샘플링 반복 적용되고있다.

의식이나 마취 기니피그 하나로 비 종단 생체 채혈 기법 개의 상이한 접근법이 제시되었다.도 1 기니피그 가능한 천자 사이트에서 횡 복재 및 족부 혈관의 과정을 도시한다. 기니 돼지 귀의 등쪽 표면에 보이는 귀 정맥은 단지 매우 소량이 필요할 때 이러한 정맥 혈액 샘플링을 위해 사용될 수있는도 2에 도시되어있다. 경정맥 혈액 샘플링을위한 기니피그의 위치는...

Watch this Scientific Journal Video about Non-Terminal Blood Sampling Techniques in Guinea Pigs at JoVE.com

Non-Terminal Blood Sampling Techniques in Guinea Pigs

알려진 모델 있지만, 기니 피그는 현재 실험 동물 과학 분야의 틈새 시장을 대표하는 제한된 데이터는 대부분의 절차의 실행에 사용할 수 있습니다. 여기에서 우리는 의식이나 마취 기니피그 중 생체 내 혈액 샘플링 기술의 비 터미널에 네 가지 방법을 제시한다.

기니 돼지의 비 터미널 혈액 샘플링 기법

Guinea pigs possess several biological similarities to humans and are validated experimental animal models1-3. However, the use of guinea pigs currently ...

non-terminal-blood-sampling-techniques-in-guinea-pigs

Research

JoVE Journal

Biology

37.3K 조회수.  University of Copenhagen.  알려진 모델 있지만, 기니 피그는 현재 실험 동물 과학 분야의 틈새 시장을 대표하는 제한된 데이터는 대부분의 절차의 실행에 사용할 수 있습니다. 여기에서 우리는 의식이나 마취 기니피그 중 생체 내 혈액 샘플링 기술의 비 터미널에 네 가지 방법을 제시한다. 

비디오: Non-Terminal Blood Sampling Techniques in Guinea Pigs

Seawater Sampling and Collection

This video documents methods for collecting coastal marine water samples and processing them for various downstream applications including biomass concentration, nucleic acid purification, cell abundance, nutrient and trace gas analyses. For today's demonstration samples were collected from the deck of the HMS John Strickland operating in Saanich Inlet. An A-frame derrick, with a multi-purpose winch and cable system, is used in combination with Niskin or Go-Flo water sampling bottles. Conductivity, Temperature, and Depth (CTD) sensors are also used to sample the underlying water mass. To minimize outgassing, trace gas samples are collected first.  Then, nutrients, water chemistry, and cell counts are determined. Finally, waters are collected for biomass filtration. The set-up and collection time for a single cast is ~1.5 hours at a maximum depth of 215 meters. Therefore, a total of 6 hours is generally needed to complete the collection series described here.

This video documents methods for collecting coastal marine water samples and processing them for various downstream applications including biomass concentration, nucleic acid purification, cell abundance, nutrient and trace gas analyses.

해수 샘플링 및 수집

This video documents methods for collecting coastal marine water samples and processing them for various downstream applications including biomass ...

연구

생물학

Optical Recording of Electrical Activity in Guinea-pig Enteric Networks using Voltage-sensitive Dyes

The enteric nervous system (ENS) is a self-contained network with identified functions, capable of performing complex behaviors in isolation. Its neurons (10 to 25 &mu;m in diameter) are arranged in plexuses that are confined to distinct planes of the gut wall 1; the myenteric plexus can be found between the longitudinal and circular muscle layers, and the submucous plexus between the circular muscle layer and the mucosa. Since the effector systems for these plexuses (transporting epithelium, endocrine cells, immune elements, blood vessels and smooth muscle) are also contained within the gut wall, semi-intact preparations can be dissected that preserve individual components of different reflex pathways. The behavior of the effector systems is controlled by the submucous and myenteric plexuses acting in concert. Therefore, detailed knowledge of synaptic interactions within and between ganglia, and of communication between the plexuses, is essential for understanding normal gastrointestinal function. The ENS, as an intact nervous system, is a unique experimental model in which one can correlate molecular and cellular events with the electrical behavior of the neuronal network and its physiological outputs. Because of the quasi-two-dimensional organization of its plexuses, the ENS is particularly well suited for the study of neural networks using multiple site optical recording techniques that employ voltage-sensitive dyes 2,7,8,9. We will illustrate here the use of a relatively new naphthylstyryl-pyridinium dye (di-4-ANEPPDHQ) 3 that offers multiple advantages over its predecessors, including very low phototoxicity, slow rate of internalization, and remarkable chemical stability. When used in conjunction with a camera that permits sub-millisecond time resolution, this dye allows us to monitor the electrical activity of all the neurons in the field of view with a maximal spatial resolution of ~ 2.5 &mu;m at 100X magnification. At lower magnification (10X or 20X), the sacrifice of single-cell resolution is compensated by a gain in perspective, revealing the intricacies of the inter-ganglionic circuitry.

This protocol illustrates how voltage-sensitive dyes enable optical recording of electrical activity from intact neural networks such as the plexuses of the guinea-pig enteric nervous system, with an adjustable resolution that ranges from single-cells to multi-ganglionic circuitry.

전압에 민감한 염료를 사용하여 기니 돼지 장용 네트워크에서 전기 활동의 광학 녹음

The enteric nervous system (ENS) is a self-contained network with identified functions, capable of performing complex behaviors in isolation. Its neurons ...

Techniques for Imaging Ca2+ Signaling in Human Sperm

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in live cells. Here we describe the method used in our laboratories for loading suspensions of human sperm with Ca2+-reporting dyes and measuring the fluorescence signal during physiological stimulation. Motile cells are isolated by direct swim-up and incubated under capacitating conditions for 0-24 h, depending upon the experiment. The cell-permeant AM (acetoxy methyl ester) ester form of the Ca2+-reporting dye is then added to a cell aliquot and a period of 1 h is allowed for loading of the dye into the cytoplasm. We use visible wavelength dyes to minimize photo-damage to the cells, but this means that ratiometric recording is not possible. Advantages and disadvantages of this approach are discussed. During the loading period cells are introduced into an imaging chamber and allowed to adhere to a poly-D-lysine coated coverslip. At the end of the loading period excess dye and loose cells are removed by connection of the chamber to the perfusion apparatus. The chamber is perfused continuously, stimuli and modified salines are then added to the perfusion header. Experiments are recorded by time-lapse acquisition of fluorescence images and analyzed in detail offline, by manually drawing regions of interest. Data are normalized to pre-stimulus levels such that, for each cell (or part of a cell), a graph showing the Ca2+ response as % change in fluorescence is obtained.

Techniques for Imaging Ca2+ Signaling in Human Sperm

Stimulus-evoked [Ca2+]i signals of individual human sperm are assessed. Motile cells are loaded with Ca2+-sensitive fluorescent dye (AM-ester method) and immobilised in a perfusable chamber. Cells are imaged by time-lapse fluorescence microscopy and stimulated via the perfusing medium. Responses of single cells (or regions) are analysed offline using Excel.

이미징 CA의 기술 2 + 인간 정자의 시그널링

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in ...

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

이온 채널 유학을위한 패치 클램프 및 재관류 기법으로 표현 Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

신생아 마우스의 폐 내피 세포의 분리 및 문화

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

Techniques for the Analysis of Extracellular Vesicles Using Flow Cytometry

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help regulate a diverse range of biological processes. Analysis of EVs using flow cytometry (FCM) has been notoriously difficult due to their small size and lack of discrete populations positive for markers of interest. Methods for EV analysis, while considerably improved over the last decade, are still a work in progress. Unfortunately, there is no one-size-fits-all protocol, and several aspects must be considered when determining the most appropriate method to use. Presented here are several different techniques for processing EVs and two protocols for analyzing EVs using either individual detection or a bead-based approach. The methods described here will assist with eliminating the antibody aggregates commonly found in commercial preparations, increasing signal&#8211;to-noise ratio, and setting gates in a rational fashion that minimizes detection of background fluorescence. The first protocol uses an individual detection method that is especially well suited for analyzing a high volume of clinical samples, while the second protocol uses a bead-based approach to capture and detect smaller EVs and exosomes.

Many different methods exist for the measurement of extracellular vesicles (EVs) using flow cytometry (FCM). Several aspects should be considered when determining the most appropriate method to use. Two protocols for measuring EVs are presented, using either individual detection or a bead-based approach.

유동 세포 계측법을 사용하여 세포 외 소포의 분석을위한 기술

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help ...

Dried Blood Spots - Preparing and Processing for Use in Immunoassays and in Molecular Techniques

The idea of collecting blood on a paper card and subsequently using the dried blood spots (DBS) for diagnostic purposes originated a century ago. Since then, DBS testing for decades has remained predominantly focused on the diagnosis of infectious diseases especially in resource-limited settings or the systematic screening of newborns for inherited metabolic disorders and only recently have a variety of new and innovative DBS applications begun to emerge. For many years, pre-analytical variables were only inappropriately considered in the field of DBS testing and even today, with the exception of newborn screening, the entire pre-analytical phase, which comprises the preparation and processing of DBS for their final analysis has not been standardized. Given this background, a comprehensive step-by-step protocol, which covers al the essential phases, is proposed, i.e., collection of blood; preparation of blood spots; drying of blood spots; storage and transportation of DBS; elution of DBS, and finally analyses of DBS eluates. The effectiveness of this protocol was first evaluated with 1,762 coupled serum/DBS pairs for detecting markers of hepatitis B virus, hepatitis C virus, and human immunodeficiency virus infections on an automated analytical platform. In a second step, the protocol was utilized during a pilot study, which was conducted on active drug users in the German cities of Berlin and Essen.

The preparing and processing of dried blood spots (DBS) for their final analysis are still poorly standardized for most diagnostic applications. To overcome this shortcoming, a comprehensive step-by-step protocol is suggested and subsequently evaluated with regard to its effectiveness for detecting markers of viral infections.

말린된 피 명소-준비 및 Immunoassays 및 분자 기술 사용에 대 한 처리

The idea of collecting blood on a paper card and subsequently using the dried blood spots (DBS) for diagnostic purposes originated a century ago. Since ...

Sampling Blood from the Lateral Tail Vein of the Rat

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, proteins, and glucose, yet the composition of the blood is dynamically regulated and easily perturbed. One factor that can change the blood composition is the stress response triggered by the sampling procedure, which can contribute to variability in the measures of interest. Here we describe a procedure for blood sampling from the lateral tail vein in the rat. This procedure offers significant advantages over other more commonly used techniques. It permits rapid sampling with minimal pain or invasiveness, without anesthesia or analgesia. Additionally, it can be used to obtain large volume samples (upwards of 1 ml in some rats), and it may be used repeatedly across experimental days. By minimizing the stress response and pain resulting from blood sampling, measures can more accurately reflect the true basal state of the animal, with minimal influence from the sampling procedure itself.

Blood samples are useful for assessing biomarkers of physiological states or disease in vivo. Here we describe the methodology to sample blood from the lateral tail vein in the rat. This method provides rapid samples with minimal pain and invasiveness.

쥐의 측면 꼬리 정맥에서 혈액을 샘플링

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, ...

Micron-scale Phenotyping Techniques of Maize Vascular Bundles Based on X-ray Microcomputed Tomography

It is necessary to accurately quantify the anatomical structures of maize materials based on high-throughput image analysis techniques. Here, we provide a 'sample preparation protocol' for maize materials (i.e., stem, leaf, and root) suitable for ordinary microcomputed tomography (micro-CT) scanning. Based on high-resolution CT images of maize stem, leaf, and root, we describe two protocols for the phenotypic analysis of vascular bundles: (1) based on the CT image of maize stem and leaf, we developed a specific image analysis pipeline to automatically extract 31 and 33 phenotypic traits of vascular bundles; (2) based on the CT image series of maize root, we set up an image processing scheme for the three-dimensional (3-D) segmentation of metaxylem vessels, and extracted two-dimensional (2-D) and 3-D phenotypic traits, such as volume, surface area of metaxylem vessels, etc. Compared with traditional manual measurement of vascular bundles of maize materials, the proposed protocols significantly improve the efficiency and accuracy of micron-scale phenotypic quantification.

We provide a novel method to improve the X-ray absorption contrast of maize tissue suitable for ordinary microcomputed tomography scanning. Based on CT images, we introduce a set of image-processing workflows for different maize materials to effectively extract microscopic phenotypes of vascular bundles of maize.

X 선 Microcomputed 단층 촬영에 따라 옥수수 혈관 번들의 미크론 단위 형질 기법

It is necessary to accurately quantify the anatomical structures of maize materials based on high-throughput image analysis techniques. Here, we provide a ...

Gonadectomy and Blood Sampling Procedures in the Small Size Teleost Model Japanese Medaka (Oryzias latipes)

Sex steroids, produced by the gonads, play an essential role in brain and pituitary tissue plasticity and in the neuroendocrine control of reproduction in all vertebrates by providing feedback to the brain and pituitary. Teleost fishes possess a higher degree of tissue plasticity and variation in reproductive&#160;strategies compared to mammals and appear to be useful models to investigate the role of sex steroids and the mechanisms by which they act. The removal of the main source of sex steroid production using gonadectomy together with blood sampling to measure steroid levels has been well-established and fairly feasible in bigger fish and is a powerful technique to investigate the role and effects of sex steroids. However, these techniques raise challenges when implemented in small size teleost models. Here, we describe the step-by-step procedures of gonadectomy in both males and female Japanese medaka followed by blood sampling. These protocols are shown to be highly feasible in medaka indicated by a high survival rate, safety for the life span and phenotype of the fish, and reproducibility in terms of sex steroid clearance. The use of these procedures combined with the other advantages of using this small teleost model will greatly improve the understanding of feedback mechanisms in the neuroendocrine control of reproduction and tissue plasticity provided by sex steroids in vertebrates.

Gonadectomy and Blood Sampling Procedures in the Small Size Teleost Model Japanese Medaka Oryzias latipes

The article describes a quick protocol to gonadectomize and sample blood from the small teleost fish, using Japanese medaka (Oryzias latipes) as a model, to investigate the role of sex steroids in animal physiology.

작은 크기의 텔레오스 모델 일본어 메다카(오리지아스 latipes)에서고나데토미및 혈액 샘플링 절차

Sex steroids, produced by the gonads, play an essential role in brain and pituitary tissue plasticity and in the neuroendocrine control of reproduction in ...