이 작품은 빅토리아 정부의 OIS 프로그램에 의한 국민 건강과 호주의 의료 연구 협의회 (NHMRC) (526615 및 545906)에서와 일부 기금에 의해 지원되었다. DS는 NHMRC의 동료입니다.

1. [3 H]-콜레스테롤의 작성 <ol><li> 연기 후드에서 1.5 ML microfuge 튜브 (0.5 잘 인당 μCi (19 kBq)은 전형적인 분석이 필요합니다)에 [3 H] 콜레스테롤 필요한 금액을 분배. </li><li> [3 H]는-콜레스테롤은 톨루엔에 정지하는 경우, 1 μCi의 최종 농도 100 %의 에탄올과 N2 가스와 resuspend (물론 37 kBq / μl. 소용돌이와 혼합으로 완전히 내려 건조. </li></ol> 2. 도금 셀 및 Labelling 세포 콜레스테롤 인간 monocytes 4,5, THP-1 인간 단핵구-macrophages 6,7,8, RAW 264.7 murine macrophages 5,9,10,11, HELA 세포 12, 내피 인간 제대 정맥 :이 프로토콜은 다음과 같은 셀 유형을 사용하여 테스트되었습니다 혈소판의 세포 (HUVEC), BHK-21 세포 9, 인간과 마우스 섬유아 세포 13,14, HepG2 인간 hepatocarcinoma 세포 15, 수정된 형태로, &lt;&gt; 16 저녁을 먹다. <ol><li> 세포를 Resuspend 그들을 계산합니다. 0.9 ML 전체 매체에서 잘 당 0.2x10 6 세포의 최종 밀도를 12 - 웰 플레이트에 플레이트 세포. 세포는 성장을 계속하고 유출 실험 시간에 잘 당 0.8x10 6 세포에 도달할 것입니다. 이러한 차별화된 THP-1 세포와 같은 분열하지 않는 세포, 내용 시딩 밀도는 당 잘 0.8x10 6 셀이 있어야합니다. 각각 또는 24 - 잘 접시를 두 번 혹은 잘 당 세포의 절반 번호 - 6을 사용하는 경우. 우물의 숫자는 각 실험 조건에 대한 네 겹의 결정하기에 충분해야합니다. </li><li> THP-1 세포의 분화가 필요한 경우, 48-72 H 위해 미디어에 PMA를 (최종 농도 0.1 μg / ml) 쇼핑 추가할 수 있습니다. </li><li> [3 H] 콜레스테롤을 포함 microfuge 튜브로 미디어의 작은 나누어지는을 추가합니다. </li><li> 완전한 미디어 별도 나누어지는 (100 μl / 음)에 [H 3] 콜레스테롤을 추가하기 전에 미디어와 튜브의 측면을 씻으십시오. </li> &lt;리&gt; 전지 (물론 당 최종 부피 1 ML 임) 우물에 [3 H] 콜레스테롤을 포함하는 미디어를 추가합니다. 세포에 대한 치료가 라벨 전에 필요하지 않은 경우 전지는 [3 H] 콜레스테롤 함유 매체에 도금 수 있습니다. </li><li> (37 ° C, 5 % CO 2) 세포 문화 인큐베이터에서 48 시간 동안 세포를 품어. </li></ol> 3. 평균 보육 <ol><li> 48시간 보육 후, 현미경으로 세포를 확인합니다. 그들은 건강과 약 80 %의 합류점에있다 확인하십시오. </li><li> 미디어는 [3 H] 콜레스테롤을 포함 제거합니다. PBS로 세포를 부드럽게 씻는다. 3 세척 번 반복합니다. </li><li> 혈청 프리 미디어를 준비합니다. 혈청 자유 언론, 필요한 경우, LXR의 작용제 (예 : 1-4 mMol / L에서 TO-901317) 또는 캠프 (단지 마우스 원산지 전지 0.3 μMol / L)를 추가하여 세포를 활성화합니다. </li><li> 각도 500 μl 혈청 자유로운 미디어를 추가합니다. </li><li> (37 ° C, 5 % CO 2) 세포 문화 인큐베이터에서 18 시간 동안 알을 품다. </li></ol> &lt;P 클래스 = &quot;jove_title&quot;&gt; 4. 콜레스테롤 유출 보육 <ol><li> 혈청이없는 배지 18 시간 배양 후 현미경으로 세포를 확인합니다. 그들이 (최소 80% confluency) 건강하고 합류있다 확인하십시오. </li><li> 혈청이없는 배지에서 콜레스테롤 유출 acceptors의 솔루션을 준비합니다. acceptors의 예는 다음과 같습니다 Apolipoprotein AI (apoA-I) (최종 농도 10 μg / ml) 쇼핑 고밀도 지방 단백질 (HDL) (최종 농도 20 μg / ml) 쇼핑 사이클로 덱스트린 (최종 농도 200 μg / ml) 쇼핑 플라즈마 (최종 농도 1~2%) </li><li> PBS로 세포를 부드럽게 씻는다. </li><li> 각 잘으로 acceptors과 혈청이없는 미디어 250 μl를 추가합니다. </li><li> 배경 유출을 (NO 수용체와 혈청 무료 미디어에 유출)를 결정 우물 한 세트를 남겨주세요 </li><li> 세포 문화 인큐베이터 2 시간 동안 세포를 (37 ° C, 5 % CO 2) 부화. 필요한 경우 유출 부화 기간은 30 분에서 8 H로 달라질 수 있습니다. </li></ol> 5. 처리 사mples <ol><li> 2 시간 후에 부화는 현미경으로 세포를 확인합니다. </li><li> 1.5 ML의 microfuge 튜브로 미디어를 수집합니다. 세포 파편을 제거하기 위해 상온에서 1-10 분 14,000 rpm으로 봐. </li><li> 7 ML의 섬광 유리병에 100 μl 미디어를 전송합니다. 5 채소 젤 플러스의 MLS (PerkinElmer)와 와동 혼합물을 추가하십시오. 4 ° C.에 남아있는 샘플을 저장 </li><li> 30 분 동안 냉장고에서 접시를 놓습니다. 각도 500 μl DH 2 O를 추가합니다. 모든 세포가 우물의 바닥에서 뜬했는지 확인하기 위해 현미경으로 세포를 확인합니다. 여전히 자기편 경우, 4 ° C에서 하룻밤이나 자국이 우물에서 DH 2 O와 접시를 떠나 하나. </li><li> 모든 세포에서 찾아낸 후에는 세포 대단히 짧은 시간을 깨고 아래로 피펫합니다. 7 ML의 섬광 튜브에 100 μl를 전송합니다. 5 채소 젤 플러스의 MLS (PerkinElmer)와 와동 혼합물을 추가하십시오. 4 ° C.에 남아있는 접시를 저장 </li><li> 신틸레이션 계수기에 섬광 튜브를 놓습니다. [3 H]를 설명할 수에 카운터 구성DPM 단위. </li></ol> 6. 결과 Analysing <ol><li> 콜레스테롤 유출의 비율은 일반적으로 콜레스테롤의 비율이 세포의 수용체로 이동으로 표시됩니다. 다음 수식이 사용됩니다 : </li></ol><img alt="등식 1" src="/files/ftp_upload/3810/3810eq1.jpg" /><ol start="2"><li> 구체적인 유출은 수용체 (빈)의 유무에 유출 차이로 계산됩니다. </li></ol><img alt="등식이" src="/files/ftp_upload/3810/3810eq2.jpg" /> 7. 타임 라인 <img alt="그림 1" src="/files/ftp_upload/3810/3810timeline.jpg" /> 8. 대표 결과 콜레스테롤 유출의 실험 결과의 예제는 그림에 표시됩니다. 1. 본 실험에서는 THP-1 인간 monocytes이였다 다른 acceptors로 macrophages과 콜레스테롤 유출로 차별했다테스트했습니다. 노 acceptors ( &quot;빈&quot;)와 중간에 콜레스테롤 유출은 0.79 %이었고,이 값은 불특정 유출으로 간주되었으며, 다른 값에서 빼는되었다. 인간 apoA - 나 (최종 농도가 30 μg / ml) 쇼핑에 콜레스테롤 유출은 4.75 %였다. 참조 플라즈마 샘플을 상호 실험적 변화를 모니터링하기 위해 본 실험에 포함되었습니다. 참조 샘플은 실험 다수에 걸쳐 데이터의 정상화에 사용하지만, 우리는 변화가 클 경우 분석을 반복하는 것이 필요하다고 볼 수 있습니다. 환자는 약물로 치료되었다 전후의 환자 혈장 (최종 농도 2%)가 테스트되었습니다. 그것이 환자의 약물에 콜레스테롤 유출을 지원하는 플라즈마의 용량에 부정적인 영향을 가지고 있다고 결론했다. 유출 실험 결과의 또 다른 예제는 그림에 표시됩니다. 2. 본 실험에서는 RAW 264.7 macrophages는 LXR의 작용제 TO-901317로 하룻밤 배양 (최종 농도에 의해 활성화 또는 활성화되지 않았습니다1 μmol / L) 및 인간 플라즈마 같은 샘플 (2 %)에 콜레스테롤 유출을 테스트했습니다. 그것은 LXR의 작용제와 ABC 방송 전송기의 세포 표현의 활성화는 세포 수용체로 콜레스테롤을 공개하는 세포의 용량을 증가한다는 결론되었다. <img alt="그림 1" src="/files/ftp_upload/3810/3810fig1.jpg" /> 1 그림. 다양한 acceptors로 THP-1 세포에서 콜레스테롤 유출. ​​콜레스테롤 유출의 비율은 (즉 레이블 콜레스테롤의 비율이 세포에서 특정 수용체로 이동) 빈 값의 뺄셈 후에 표시됩니다. 평균 ± 네 겹의 결정의 SD, * P &lt;0.05 있습니다. <img alt="그림 2" src="/files/ftp_upload/3810/3810fig2.jpg" /> 그림 2. 콜레스테롤 유출의 RAW 264.7 인간의 플라즈마에 LXR의 작용제로 활성화하거나 활성화하지. 비율 (즉, 레이블 콜레스테롤의 비율에서 콜레스테롤 유출은 세포로부터로 이동지정된 수용체)는 빈 값 뺄셈 후에 표시됩니다. 평균 ± 네 겹의 결정의 SD, * P &lt;0.001합니다.

<ol>
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저자들은 본 연구와 관련된 어떠한 충돌하는 이익을 선언 없습니다.

콜레스테롤 유출을 측정하기위한 설명한 방법은 세포에서 세포외 콜레스테롤 수용체에 콜레스테롤의 움직임을 측정하기 위해 설계되었습니다. 이 방법론을 이해하는 몇 가지 중요한 고려 사항이 있습니다. Labelling와 평균 설명한 방법론에 표시된 콜레스테롤이 혈청 함유 혈청에 추가됩니다. 자세히 조사한 적이 있지만, 그것은 콜레스테롤이 혈청 lipoprote...

<table border="1"><tbody><tr><td> 시약 \ 셀 </td><td> HELA </td><td> THP-1 </td><td> RAW </td><td> HUVEC </td><td> BHK-21 </td><td> HFF </td></tr><tr><td> 전체 미디어 </td><td colspan="3"> RPMI - 10 % FCS - 1퍼센트 L-글루타민 - 0.2 % 펜 strep </td><td> M199 - 5% FCS - 1퍼센트 L-글루타민 - 1 %의 펜 strep - 2 %의 HEPES (1M) - 7퍼센트 내피 세포 형성제 (ECGS) </td><td colspan="2"> DMEM - 10 % FCS - 1퍼센트 L-글루타민 - 0.2 % 펜 strep </td></tr><tr><td> 세럼 무료 미디어 </td><td colspan="3"> RPMI - 1퍼센트 L-글루타민 - 0.2 % 펜 strep </td><td> M199 - 1퍼센트 L-글루타민 - 1 %의 펜 strep - 2 %의 HEPES (1M) - 7퍼센트 내피 세포 성장일 보충 (ECGS) </td><td colspan="2"> DMEM - 1퍼센트 L-글루타민 - 0.2 % 펜 strep </td></tr><tr><td> 항로 방법 </td><td> 1 X 트립신 </td><td> 서스펜션 </td><td> 긁다 </td><td colspan="3"> 1 X 트립신 </td></tr><tr><td> 항로 비율 </td><td> 3시 10분 </td><td> 1시 3분 </td><td></td><td> 1시 20분 </td><td> 1시 3분 </td><td> 1시 3분 </td></tr><tr><td> 세포 활성화 (선택 사항) </td><td></td><td colspan="5"> TO-901317 - LXR-작용제 - 최종 conc. 4 μm의 </td></tr><tr><td> 기타 시약 </td><td></td><td> phorbol 12-myristate의 13 아세테이트 (PMA) - 세포 분화 - 최종 conc. 0.1μg/ml </td><td></td><td></td><td></td><td></td></tr></tbody></table>

cholesterol efflux assay

세포의 콜레스테롤 함량은 세포 세포막의 파괴, apoptosis과 괴사 1 셀 결과에 너무 많이 또는 너무 적게 콜레스테롤, 매우 빡빡 한도 내에서 유지되어야합니다. 세포는 세포 합성에서와 플라즈마 lipoproteins에서 소스 콜레스테롤, 두 소스가 완전히 콜레스테롤에 대한 세포 &#39;요건을 충족하기 충분합니다. 콜레스테롤 합성과 이해의 프로세스를 긴밀하게 규제하고 콜레스테롤의 결함이 드문이 있습니다. 과도한 콜레스테롤은보다 일반적인 문제 3. hepatocytes의 예외와 함께 어느 정도 부신 피질 세포로, 세포는 콜레스테롤을 저하 수 없습니다. 세포는 콜레스테롤 콘텐츠를 줄이기위한 두 가지 옵션이 있습니다 또한 독성 cholesteryl 에스테르로 세포를 오버로드로 cholesteryl 에스테르, 제한된 용량과 옵션으로 콜레스테롤을 변환하고, 콜레스테롤 유출, 잠재적으로 무제한 용량과 옵션이 있습니다. 콜레스테롤 유출은 사양입니다이러한 ATP 결합 카세트 수송 단백질 A1 (ABCA1)와 G1 (ABCG1) 및 청소 수용체 타입 B1과 같은 세포내 이동 장치의 숫자에 의해 규제되고 ific 과정입니다. 혈장의 콜레스테롤의 자연 수용체가 고밀도 지단 백질 (HDL)과 apolipoprotein AI입니다. 콜레스테롤 유출 분석은 교양 세포에서 콜레스테롤 유출의 속도를 quantitate하도록 설계되었습니다. 그것은 콜레스테롤 유출 및 / 또는 세포에서 풀려나는 콜레스테롤을 수락하기위한 플라즈마 acceptors의 용량을 유지하기 위해 세포의 용량을 측정합니다. 분석은 다음 단계로 구성되어 있습니다. 1 단계 : 혈청 함유 배지에 표시된 콜레스테롤을 추가하고 24-48 H에 대한 세포로 잠복기에 의해 세포의 콜레스테롤 레이블링. 이 단계는 콜레스테롤과 세포의로드와 결합할 수 있습니다. 2 단계 : 모든 세포내 콜레스테롤 풀 사이 라벨 콜레스테롤 평형 위해 혈청이없는 배지에서 세포의 배양. 이 단계는 셀룰러 채널의 활성화와 결합될 수도 있습니다olesterol의 전송기. 3 단계 : 세포 수용체와 세포의 수용체로 분류 콜레스테롤 움직임 quantitation과 세포의 배양. 콜레스테롤 엽 성의 전구 물질이 새롭게 합성된 콜레스테롤 레이블을 지정하는 데 사용한다면, 네번째 단계, 콜레스테롤 정화가 필요할 수 있습니다. 분석은 다음과 같은 정보를 제공합니다 :은 (i) 특정 치료 (돌연변이, 노크 다운, overexpression 또는 치료) 유출 콜레스테롤을 세포의 용량에 영향을 그리고 (ii) 방법 플라즈마 acceptors의 용량은 콜레스테롤을 수락하는 방법 질병이나 치료에 의해 영향을받습니다. 이 방법은 종종 심장 혈관 연구, 신진 대사 및 neurodegenerative 장애, 전염성 및 생식 질병의 컨텍스트에서 사용됩니다.

Watch this Scientific Journal Video about Cholesterol Efflux Assay at JoVE.com

Cholesterol Efflux Assay | Medicine | Video

Cholesterol Efflux Assay

콜레스테롤 분석은 교양 세포에서 콜레스테롤 유출의 속도와 세포에서 풀려나는 콜레스테롤을 수락하기위한 플라즈마 acceptors의 용량을 quantitate하도록 설계되었습니다. 분석은 세포 내 수영장과 세포 수용체에 콜레스테롤의 릴리스 간의 콜레스테롤 콜레스테롤, 평균으로 세포를 레이블링으로 구성되어 있습니다.

콜레스테롤 유출 분석

Cholesterol content of cells must be maintained within the very tight limits, too much or too little cholesterol in a cell results in disruption of ...

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29.5K 조회수.  Baker IDI Heart and Diabetes Institute. 콜레스테롤 분석은 교양 세포에서 콜레스테롤 유출의 속도와 세포에서 풀려나는 콜레스테롤을 수락하기위한 플라즈마 acceptors의 용량을 quantitate하도록 설계되었습니다. 분석은 세포 내 수영장과 세포 수용체에 콜레스테롤의 릴리스 간의 콜레스테롤 콜레스테롤, 평균으로 세포를 레이블링으로 구성되어 있습니다.

비디오: Cholesterol Efflux Assay

Experimental Metastasis Assay

Metastasis is the leading cause of death in cancer patients. To understand the mechanism of metastasis, an experimental metastasis assay was established using immunodeficient mice. This article delineates the procedures involved in this assay, including sample preparation, intravenous injection, and culturing cells from lung metastases. Briefly, a pre-determined number of human cancer cells were prepared in vitro and directly injected into the circulation of immunodeficient mice through their tail veins. A small number of cells survive the turbulence in the circulation and grow as metastases in internal organs, such as lung. The injected mice are dissected after a certain period. The tissue distribution of metastases is determined under a dissecting microscope. The number of metastases in a specific tissue is counted and it directly correlates with the metastatic ability of the injected cancer cells. The arisen metastases are isolated and cultured in vitro as cell lines, which often show enhanced metastatic abilities than the parental line when injected again into immunodeficient mice. These highly metastatic derivatives become useful tools for identifying genes or molecular pathways that regulate metastatic progression.

This article describes the procedures of an experimental metastasis assay that is used to determine the metastatic potential of human cancer cell lines.

실험 전이 분석

Metastasis is the leading cause of death in cancer patients.  To understand the mechanism of metastasis, an experimental metastasis assay was established ...

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의학

In Vivo Canine Muscle Function Assay

We describe a minimally-invasive and reproducible method to measure canine pelvic limb muscle strength and muscle response to repeated eccentric 
contractions. The pelvic limb of an anesthetized dog is immobilized in a stereotactic frame to align the tibia at a right angle to the femur. Adhesive wrap affixes the 
paw to a pedal mounted on the shaft of a servomotor to measure torque. Percutaneous nerve stimulation activates pelvic limb muscles of the paw to either push (extend) or 
pull (flex) against the pedal to generate isometric torque. Percutaneous tibial nerve stimulation activates tibiotarsal extensor muscles. Repeated eccentric (lengthening) 
contractions are induced in the tibiotarsal flexor muscles by percutaneous peroneal nerve stimulation. The eccentric protocol consists of an initial isometric contraction 
followed by a forced stretch imposed by the servomotor. The rotation effectively lengthens the muscle while it contracts, e.g., an eccentric contraction. During 
stimulation flexor muscles are subjected to an 800 msec isometric and 200 msec eccentric contraction. This procedure is repeated every 5 sec. To avoid fatigue, 4 min rest 
follows every 10 contractions with a total of 30 contractions performed.

In Vivo Canine Muscle Function Assay

We describe a minimally-invasive and painless method to measure canine hindlimb muscle strength and muscle response to repeated eccentric contractions.

VIVO 개 근육 기능 분석에

We describe a minimally-invasive and reproducible method to measure canine pelvic limb muscle strength and muscle response to repeated eccentric 
...

A Mouse Model of the Cornea Pocket Assay for Angiogenesis Study

A normal cornea is clear of vascular tissues. However, blood vessels can be induced to grow and survive in the cornea when potent angiogenic factors are administered 1. This uniqueness has made the cornea pocket assay one of the most used models for angiogenesis studies. The cornea composes multiple layers of cells. It is therefore possible to embed a pellet containing the angiogenic factor of interest in the cornea to investigate its angiogenic effect 2,3. Here, we provide a step by step demonstration of how to (I) produce the angiogenic factor-containing pellet (II) embed the pellet into the cornea (III) analyze the angiogenesis induced by the angiogenic factor of interest. Since the basic fibroblast growth factor (bFGF) is known as one of the most potent angiogenic factors 4, it is used here to induce angiogenesis in the cornea.

The cornea is unique in that it lacks vascular tissues. However, robust blood vessel growth and survival can be induced in the cornea by potent angiogenic factors. Therefore, the cornea can provide with us a valuable tool for angiogenic studies. This protocol demonstrates how to perform the mouse model of cornea pocket assay and how to assess the angiogenesis induced by angiogenic factors using this model.

Angiogenesis 연구에 대한 각막 포켓 분석의 마우스 모델

A normal cornea is clear of vascular tissues. However, blood vessels can be induced to grow and survive in the cornea when potent angiogenic factors are ...

Spheroid Assay to Measure TGF-&#946;-induced Invasion

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis at later stage1,2. Moreover, TGF-&beta; is frequently overexpressed in breast cancer and its expression correlates with poor prognosis and metastasis 3,4. The mechanisms by which TGF-&beta; induces invasion are not well understood.
TGF-&beta; elicits its cellular responses via TGF-&beta; type II (T&beta;RII) and type I (T&beta;RI) receptors. Upon TGF-&beta;-induced heteromeric complex formation, T&beta;RII phosphorylates the T&beta;RI. The activated T&beta;RI initiates its intracellular canonical signaling pathway by phosphorylating receptor Smads (R-Smads), i.e. Smad2 and Smad3. These activated R-Smads form heteromeric complexes with Smad4, which accumulate in the nucleus and regulate the transcription of target genes5. In addition to the previously described Smad pathway, receptor activation results in activation of several other non-Smad signaling pathways, for example Mitogen Activated Protein Kinase (MAPK) pathways6.
To study the role of TGF-&beta; in different stages of breast cancer, we made use of the MCF10A cell system. This system consists of spontaneously immortalized MCF10A1 (M1) breast epithelial cells7, the H-RAS transformed M1-derivative MCF10AneoT (M2), which produces premalignant lesions in mice8, and the M2-derivative MCF10CA1a (M4), which was established from M2 xenografts and forms high grade carcinomas with the ability to metastasize to the lung9. This MCF10A series offers the possibility to study the responses of cells with different grades of malignancy that are not biased by a different genetic background.
For the analysis of TGF-&beta;-induced invasion, we generated homotypic MCF10A spheroid cell cultures embedded in a 3D collagen matrix in vitro (Fig 1). Such models closely resemble human tumors in vivo by establishing a gradient of oxygen and nutrients, resulting in active and invasive cells on the outside and quiescent or even necrotic cells in the inside of the spheroid10. Spheroid based assays have also been shown to better recapitulate drug resistance than monolayer cultures11. This MCF10 3D model system allowed us to investigate the impact of TGF-&beta; signaling on the invasive properties of breast cells in different stages of malignancy.

Spheroid Assay to Measure TGF-&amp;#946;-induced Invasion

An assay to quantitatively measure Transforming Growth Factor (TGF)-&#x03B2;-induced invasion in 3-dimensional collagen gels is described. This assay takes advantage of the MCF10A series of cell lines, which represent different stages of breast cancer development. This method can be adopted to be used with other cell lines and might be used to investigate other potential activators or inhibitors of invasion.

TGF - β - 유발 침략을 측정하는 회전 타원체 분석

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis ...

An in vivo Assay to Test Blood Vessel Permeability

This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under physiologic conditions the endothelium is impermeable to albumin, so Evans blue bound albumin remains restricted within blood vessels. In pathologic conditions that promote increased vascular permeability endothelial cells partially lose their close contacts and the endothelium becomes permeable to small proteins such as albumin. This condition allows for extravasation of Evans Blue in tissues. A healthy endothelium prevents extravasation of the dye in the neighboring vascularized tissues. Organs with increased permeability will show significantly increased blue coloration compared to organs with intact endothelium. The level of vascular permeability can be assessed by simple visualization or by quantitative measurement of the dye incorporated per milligram of tissue of control versus experimental animal/tissue. Two powerful aspects of this assay are its simplicity and quantitative characteristics. Evans Blue dye can be extracted from tissues by incubating a specific amount of tissue in formamide. Evans Blue absorbance maximum is at 620 nm and absorbance minimum is at 740 nm. By using a standard curve for Evans Blue, optical density measurements can be converted into milligram dye captured per milligram of tissue. Statistical analysis should be used to assess significant differences in vascular permeability.

An in vivo Assay to Test Blood Vessel Permeability

We are presenting an in vivo assay to test blood vessel permeability. This assay is based on intravenous injection of a dye and subsequent visualization of its diffusion into interstitial spaces.

<em&gt; 생체 내</em혈관 투과율을 테스트하려면&gt; 분석

This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under ...

The In ovo CAM-assay as a Xenograft Model for Sarcoma

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for personalized medicine after stratification, explaining the current interest in developing a reproducible and low-cost xenotransplant model for this disease. The chick chorioallantoic membrane is a natural immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition, it is easily accessed, manipulated and imaged using optical and fluorescence stereomicroscopy. Histology further allows detailed analysis of heterotypic cellular interactions.
This protocol describes in detail the in ovo grafting of the chorioallantoic membrane with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines (Saos-2 and SW1353). The chick survival rates are up to 75%. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior. For localized grafting of single cell suspensions, ECM gel provides significant advantages over inert containment materials. The Ki67 proliferation index is related to the distance of the cells from the surface of the CAM and the duration of application on the CAM, the latter determining a time frame for the addition of therapeutic products.

The In ovo CAM-assay as a Xenograft Model for Sarcoma

The in ovo chorioallantoic membrane (CAM) is grafted with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior.

<em&gt; OVO에서</em육종을위한 Xenograftmodel으로&gt; CAM-분석

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for ...

Simulating Pancreatic Neuroplasticity: In Vitro Dual-neuron Plasticity Assay

Neuroplasticity is an inherent feature of the enteric nervous system and gastrointestinal (GI) innervation under pathological conditions. However, the pathophysiological role of neuroplasticity in GI disorders remains unknown. Novel experimental models which allow simulation and modulation of GI neuroplasticity may enable enhanced appreciation of the contribution of neuroplasticity in particular GI diseases such as pancreatic cancer (PCa) and chronic pancreatitis (CP). Here, we present a protocol for simulation of pancreatic neuroplasticity under in vitro conditions using newborn rat dorsal root ganglia (DRG) and myenteric plexus (MP) neurons. This dual-neuron approach not only permits monitoring of both organ-intrinsic and -extrinsic neuroplasticity, but also represents a valuable tool to assess neuronal and glial morphology and electrophysiology. Moreover, it allows functional modulation of supplied microenvironmental contents for studying their impact on neuroplasticity. Once established, the present neuroplasticity assay bears the potential of being applicable to the study of neuroplasticity in any GI organ.

Simulating Pancreatic Neuroplasticity: In Vitro Dual-neuron Plasticity Assay

Neuronal plasticity is an increasingly recognized, but insufficiently understood feature of the gastrointestinal (GI) tract. Here, in the example of human pancreatic disorders, we present an in vitro neuroplasticity assay for the study of neuronal plasticity in the GI tract at both morphological and functional level.

췌장 신경 가소성 시뮬레이션 :<em&gt; 체외</em&gt; 이중 신경 가소성 분석

Neuroplasticity is an inherent feature of the enteric nervous system and gastrointestinal (GI) innervation under pathological conditions. However, the ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

3 차원 (3D) 종양 회전 타원체 침공 분석

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

Differential Effects of Lipid-lowering Drugs in Modulating Morphology of Cholesterol Particles

Treatment of dyslipidemia patients with lipid-lowering drugs leads to a significant reduction in low-density lipoproteins (LDL) level and a low to moderate level of increase in high-density lipoprotein (HDL) cholesterol in plasma. However, a possible role of these drugs in altering morphology and distribution of cholesterol particles is poorly understood. Here, we describe the in vitro evaluation of lipid-lowering drug effects in modulating morphological features of cholesterol particles using the plaque array method in combination with imaging flow cytometry. Image analyses of the cholesterol particles indicated that lovastatin, simvastatin, ezetimibe, and atorvastatin induce the formation of both globular and linear strand-shaped particles, whereas niacin, fibrates, fluvastatin, and rosuvastatin induce the formation of only globular-shaped particles. Next, purified very low-density lipoprotein (VLDL) and LDL particles incubated with these drugs showed changes in the morphology and image texture of cholesterol particles subpopulations. Furthermore, screening of 50 serum samples revealed the presence of a higher level of linear shaped HDL cholesterol particles in subjects with dyslipidemia (mean of 18.3%) compared to the age-matched normal (mean of 11.1%) samples. We also observed considerable variations in lipid-lowering drug effects on reducing linear shaped LDL and HDL cholesterol particles formation in serum samples. These findings indicate that lipid-lowering drugs, in addition to their cell-mediated hypolipidemic effects, may directly modulate morphology of cholesterol particles by a non-enzymatic mechanism of action. The outcomes of these results have potential to inform diagnosis of atherosclerosis and predict optimal lipid-lowering therapy.

The objective of this study was to evaluate in vitro lipid-lowering drug effects in modulating the morphology of cholesterol particles. Comparison of lipid-lowering drugs revealed variations in their effect in modulating the morphological features of cholesterol particles.

콜레스테롤 입자의 형태를 변조에 지질 저하 약물의 차동 효과

Treatment of dyslipidemia patients with lipid-lowering drugs leads to a significant reduction in low-density lipoproteins (LDL) level and a low to ...

High-throughput Nitrobenzoxadiazole-labeled Cholesterol Efflux Assay

Atherosclerosis leads to cardiovascular disease (CVD). It is still unclear whether cholesterol-HDL (cHDL) concentration plays a causal role in atherosclerosis development. However, an important factor in early stages of atheroma plaque formation is cholesterol efflux capacity to HDL (the ability of HDL particles to accept cholesterol from macrophages) in order to avoid foam cell formation. This is a key step in avoiding the accumulation of cholesterol in the endothelium and a part of reverse cholesterol transport (RCT) to eliminate cholesterol through the liver. Cholesterol efflux capacity to serum or plasma in macrophage cell models is a promising tool that can be used as biomarker for atherosclerosis. Traditionally, [3H]-cholesterol has been used in cholesterol efflux assays. In this study, we aim to develop a safer and faster strategy using fluorescent labelled-cholesterol (NBD-cholesterol) in a cellular assay to trace the cholesterol uptake and efflux process in THP-1-derived macrophages. Finally, we optimize and standardize the NBD-cholesterol efflux method and develop a high-throughput analysis using 96-well plates.

Measurement of in vitro cholesterol efflux capacity to serum or plasma in macrophage cell models is a promising tool as a biomarker for atherosclerosis. In the present study, we optimize and standardize a fluorescent NBD-cholesterol efflux method and develop a high-throughput analysis using 96-well plates.