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

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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29.8K 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

Enrichment of Mammalian Tissues and Xenopus Oocytes with Cholesterol

Cholesterol enrichment of mammalian tissues and cells, including Xenopus oocytes used for studying cell function, can be accomplished using a variety of methods. Here, we describe two important approaches used for this purpose. First, we describe how to enrich tissues and cells with cholesterol using cyclodextrin saturated with cholesterol using cerebral arteries (tissues) and hippocampal neurons (cells) as examples. This approach can be used for any type of tissue, cells, or cell lines. An alternative approach for cholesterol enrichment involves the use of low-density lipoprotein (LDL). The advantage of this approach is that it uses part of the natural cholesterol homeostasis machinery of the cell. However, whereas the cyclodextrin approach can be applied to enrich any cell type of interest with cholesterol, the LDL approach is limited to cells that express LDL receptors (e.g., liver cells, bone marrow-derived cells such as blood leukocytes and tissue macrophages), and the level of enrichment depends on the concentration and the mobility of the LDL receptor. Furthermore, LDL particles include other lipids, so cholesterol delivery is nonspecific. Second, we describe how to enrich Xenopus oocytes with cholesterol using a phospholipid-based dispersion (i.e., liposomes) that includes cholesterol. Xenopus oocytes constitute a popular heterologous expression system used for studying cell and protein function. For both the cyclodextrin-based cholesterol enrichment approach of mammalian tissue (cerebral arteries) and for the phospholipid-based cholesterol enrichment approach of Xenopus oocytes, we demonstrate that cholesterol levels reach a maximum following 5 min of incubation. This level of cholesterol remains constant during extended periods of incubation (e.g., 60 min). Together, these data provide the basis for optimized temporal conditions for cholesterol enrichment of tissues, cells, and Xenopus oocytes for functional studies aimed at interrogating the impact of cholesterol enrichment.

Enrichment of Mammalian Tissues and Xenopus Oocytes with Cholesterol

Two methods of cholesterol enrichment are presented: the application of cyclodextrin saturated with cholesterol to enrich mammalian tissues and cells, and the use of cholesterol-enriched phospholipid-based dispersions (liposomes) to enrich Xenopus oocytes. These methods are instrumental for determining the impact of elevated cholesterol levels in molecular, cellular, and organ function.

포유류 조직과 콜레스테롤을 함유한 제노푸스 우세포의 농축

Cholesterol enrichment of mammalian tissues and cells, including Xenopus oocytes used for studying cell function, can be accomplished using a variety of ...

연구

생화학

Spot Variation Fluorescence Correlation Spectroscopy for Analysis of Molecular Diffusion at the Plasma Membrane of Living Cells

Dynamic biological processes in living cells, including those associated with plasma membrane organization, occur on various spatial and temporal scales, ranging from nanometers to micrometers and microseconds to minutes, respectively. Such a broad range of biological processes challenges conventional microscopy approaches. Here, we detail the procedure for implementing spot variation Fluorescence Correlation Spectroscopy (svFCS) measurements using a classical fluorescence microscope that has been customized. The protocol includes a specific performance check of the svFCS setup and the guidelines for molecular diffusion measurements by svFCS on the plasma membrane of living cells under physiological conditions. Additionally, we provide a procedure for disrupting plasma membrane raft nanodomains by cholesterol oxidase treatment and demonstrate how these changes in the lateral organization of the plasma membrane might be revealed by svFCS analysis. In conclusion, this fluorescence-based method can provide unprecedented details on the lateral organization of the plasma membrane with the appropriate spatial and temporal resolution.

This article aims to present a protocol on how to build a spot variation Fluorescence Correlation Spectroscopy (svFCS) microscope to measure molecular diffusion at the plasma membrane of living cells.

살아있는 세포의 원형질막에서의 분자 확산 분석을위한 스팟 변이 형광 상관 분광법

Dynamic biological processes in living cells, including those associated with plasma membrane organization, occur on various spatial and temporal scales, ...

Cell-free Biochemical Fluorometric Enzymatic Assay for High-throughput Measurement of Lipid Peroxidation in High Density Lipoprotein

Low high-density lipoprotein cholesterol (HDL-C) levels are one of the most powerful independent negative predictors of atherosclerotic cardiovascular disease (CVD). The structure and function of HDL rather than HDL-C may more accurately predict atherosclerosis. Several HDL protein and lipid compositional changes that impair HDL function occur in inflammatory states such as atherosclerosis. HDL function is usually determined by cell based assays such as cholesterol efflux assay but these assays have numerous drawbacks lack of standardization. Cell-free assays may give more robust measures of HDL function compared to cell-based assays. HDL oxidation impairs HDL function. HDL has a major role in lipid peroxide transport and high amount of lipid peroxides is related to abnormal HDL function. Lipid-probe interactions should be considered when interpreting the results of non-enzymatic fluorescence assays for measuring the lipid oxidative state. This motivated us to develop a cell-free biochemical enzymatic method to assess HDL lipid peroxide content (HDLox) that contributes to HDL dysfunction. This method is based on the enzyme horseradish peroxidase (HRP) and the fluorochrome Amplex Red that can quantify (without cholesterol oxidase) the lipid peroxide content per mg of HDL-C. Here a protocol is describedfor determination of HDL-lipid peroxidation using the fluorochrome reagent. Assay variability can be reduced by strict standardization of experimental conditions. Higher HDLox values are associated with reduced HDL antioxidant function. The readout of this assay is associated with readouts of validated cell-based assays, surrogate measures of cardiovascular disease, systemic inflammation, immune dysfunction, and associated cardiovascular and metabolic risk phenotypes. This technical approach is a robust method to assess HDL function in human disease where systemic inflammation, oxidative stress and oxidized lipids have a key role (such as atherosclerosis).

We describe here a fluorometric cell-free biochemical assay for determination of HDL-lipid peroxidation. This rapid and reproducible assay can be used to determine HDL function in large scale studies and can contribute to our understanding of HDL function in human disease.

고밀도 지단백에 지질 과산화의 높은 처리량 측정을 위한 생 화 확 적인 세포-무료 Fluorometric 효소 분석 결과

Low high-density lipoprotein cholesterol (HDL-C) levels are one of the most powerful independent negative predictors of atherosclerotic cardiovascular ...

면역학 및 감염병학

Lipid Exchange Assay in Living Cells

Lipid rafts are dynamic, ordered domains in the plasma membrane often formed during membrane protein clustering and signaling. The lipid identity of the outer leaflet drives the membrane&#39;s propensity to form lipid rafts. The transient nature of lipid rafts makes it difficult to study in living cells. Therefore, methods that add or remove raft-forming lipids at&#160;the outer leaflet of living cells facilitate studying the characteristics of rafts, such as their effects on membrane proteins. Lipid exchange experiments developed in our lab utilize lipid-loaded cyclodextrins to remove and add exogenous phospholipids to change the lipid constitution of the plasma membrane. Substituting the membrane with a raft or non-raft-forming lipid can aid in studying the effects on transmembrane protein activity. Here, we describe a method for lipid exchange on the outer leaflet of the plasma membrane using lipid-loaded cyclodextrin. We demonstrate the preparation of the exchange media and the subsequent treatment of attached mammalian cells. We also showcase how to measure the efficiency of exchange using HP-TLC. This protocol yields a nearly complete replacement of the outer leaflet with exogenous lipids without altering cellular viability, permitting further experimentation on modified intact plasma membranes.

We describe a method for using cyclodextrin to mediate exchange between lipids of the plasma membrane with exogenous lipids. This technique can be paired with experiments studying transmembrane proteins, which behave differently in lipid raft-like environments than they do in non-raft-like environments.

살아있는 세포의 지질 교환 분석

Lipid rafts are dynamic, ordered domains in the plasma membrane often formed during membrane protein clustering and signaling. The lipid identity of the ...

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 ...

의학

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.

높은 처리량 Nitrobenzoxadiazole 표시 된 콜레스테롤 경과 분석 결과

Atherosclerosis leads to cardiovascular disease (CVD). It is still unclear whether cholesterol-HDL (cHDL) concentration plays a causal role in ...

A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters

The last 15 years have been characterized by an explosion in the ability to overexpress and purify membrane proteins from prokaryotic organisms as well as from eukaryotes. This increase has been largely driven by the successful push to obtain structural information on membrane proteins. However, the ability to functionally interrogate these proteins has not advanced at the same rate and is often limited to qualitative assays of limited quantitative value, thereby limiting the mechanistic insights that they can provide. An assay to quantitatively investigate the transport activity of reconstituted Cl- channels or transporters is described. The assay is based on the measure of the efflux rate of Cl- from proteoliposomes following the addition of the K+ ionophore valinomycin to shunt the membrane potential. An ion sensitive electrode is used to follow the time-course of ion efflux from proteoliposomes reconstituted with the desired protein. The method is highly suited for mechanistic studies, as it allows for the quantitative determination of key properties of the reconstituted protein, such as its unitary transport rate, the fraction of active protein and the molecular mass of the functional unit. The assay can also be utilized to determine the effect of small molecule compounds that directly inhibit/activate the reconstituted protein, as well as to test the modulatory effects of the membrane composition or lipid-modifying reagents. Where possible, direct comparison between results obtained using this method were found to be in good agreement with those obtained using electrophysiological approaches. The technique is illustrated using CLC-ec1, a CLC-type H+/Cl- exchanger, as a model system. The efflux assay can be utilized to study any Cl- conducting channel/transporter and, with minimal changes, can be adapted to study any ion-transporting protein.

A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters

Proteoliposomes are used to study purified channels and transporters reconstituted in a well-defined biochemical environment. An experimental procedure to measure efflux mediated by these proteins is illustrated. The steps to prepare proteoliposomes, perform the recordings, and analyze data to quantitatively determine the functional properties of the reconstituted protein are described.

Proteoliposome 기반 유출 분석은 CL의 단일 분자의 속성을 확인하는<sup&gt; -</sup&gt; 채널 및 전송기

The last 15 years have been characterized by an explosion in the ability to overexpress and purify membrane proteins from prokaryotic organisms as well as ...

생물학

Using Caco-2 Cells to Study Lipid Transport by the Intestine

Studies of dietary fat absorption are generally conducted by using an animal model equipped with a lymph cannula. Although this animal model is widely accepted as the in vivo model of dietary fat absorption, the surgical techniques involved are challenging and expensive. Genetic manipulation of the animal model is also costly and time consuming. The alternative in vitro model is arguably more affordable, timesaving, and less challenging. Importantly, the in vitro model allows investigators to examine the enterocytes as an isolated system, reducing the complexity inherent in the whole organism model. This paper describes how human colon carcinoma cells (Caco-2) can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs and vitamins. It explains the proper maintenance of Caco-2 cells and the preparation of their lipid mixture; and it further discusses the valuable option of using the permeable membrane system. Since differentiated Caco-2 cells are polarized, the main advantage of using the permeable membrane system is that it separates the apical from the basolateral compartment. Consequently, the lipid mixture can be added to the apical compartment while the lipoproteins can be collected from the basolateral compartment. In addition, the effectiveness of the lentivirus expression system in upregulating gene expression in Caco-2 cells is discussed. Lastly, this paper describes how to confirm the successful isolation of intestinal lipoproteins by transmission electron microscopy (TEM).

Caco-2 cells can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs/vitamins. The permeable membrane system separates the apical from the basolateral compartment, while the lentivirus expression system offers an effective gene overexpression method. The isolation of lipoproteins is confirmed by TEM.

소장에 의해 지질 전송을 연구하기 위해 카코 2 셀을 사용하여

Studies of dietary fat absorption are generally conducted by using an animal model equipped with a lymph cannula. Although this animal model is widely ...

LDL Cholesterol Uptake Assay Using Live Cell Imaging Analysis with Cell Health Monitoring

The regulation of LDL cholesterol uptake through LDLR-mediated endocytosis is an important area of study in various major pathologies including metabolic disorder, cardiovascular disease, and kidney disease. Currently, there is no available method to assess LDL uptake while simultaneously monitoring for health of the cells. The current study presents a protocol, using a live cell imaging analysis system, to acquire serial measurements of LDL influx with concurrent monitoring for cell health. This novel technique is tested in three human cell lines (hepatic, renal tubular epithelial, and coronary artery endothelial cells) over a four-hour time course. Moreover, the sensitivity of this technique is validated with well-known LDL uptake inhibitors, Dynasore and recombinant PCSK9 protein, as well as by an LDL uptake promoter, Simvastatin. Taken together, this method provides a medium-to-high throughput platform for simultaneously screening pharmacological activity as well as monitoring of cell morphology, hence cytotoxicity of compounds regulating LDL influx. The analysis can be used with different imaging systems and analytical software.

This protocol provides an efficient approach to measuring LDL cholesterol uptake with real time influx rates using a live cell imaging system in various cell types. This technique provides a platform to screen the pharmacological activity of compounds affecting LDL influx while monitoring for cell morphology and hence potential cytotoxicity.

LDL 콜레스테롤 이해 분석 결과 라이브 셀 이미징 분석 셀을 사용 하 여 상태 모니터링

The regulation of LDL cholesterol uptake through LDLR-mediated endocytosis is an important area of study in various major pathologies including metabolic ...

The Isolation of Flowing Mesenteric Lymph in Mice to Quantify In Vivo Kinetics of Dietary Lipid Absorption and Chylomicron Secretion

Intestinal lipoproteins, especially triglyceride-rich chylomicrons, are a major driver of metabolism, inflammation, and cardiovascular diseases. However, isolating intestinal lipoproteins is very difficult in vivo because they are first secreted from the small intestine into the mesenteric lymphatics. Chylomicron-containing lymph then empties into the subclavian vein from the thoracic duct to deliver components of the meal to the heart, lungs, and, ultimately, whole-body circulation. Isolating na&#239;ve chylomicrons is impossible from blood since chylomicron triglyceride undergoes hydrolysis immediately upon interaction with lipoprotein lipase and other lipoprotein receptors in circulation. Therefore, the original 2-day lymph fistula procedure, described by Bollman et al. in rats, has historically been used to isolate fresh mesenteric lymph before its entry into the thoracic vein. That protocol has been improved upon and professionalized by the laboratory of Patrick Tso for the last 45 years, allowing for the analysis of these critical lipoproteins and secretions from the gut. The Tso lymph fistula procedure has now been updated and is presented here visually for the first time. This revised procedure is a single-day surgical technique for installing a duodenal feeding tube, cannulating the mesenteric lymph duct, and collecting lymph after a meal in conscious mice. The major benefits of these new techniques include the ability to reproducibly collect lymph from mice (which harnesses the power of genetic mouse models); the reduced anesthesia time for mice during the implantation of the duodenal infusion tube and the lymph cannula; the ability to continuously sample lymph throughout the feeding and post-prandial period; the ability to quantitatively measure hormones and cytokines before their dilution and enzymatic hydrolysis in blood; and the ability to collect large quantities of lymph for isolating intestinal lipoproteins. This technique is a powerful tool for directly and quantitatively measuring dietary nutrient absorption, intestinal lipoprotein synthesis, and chylomicron secretion.

The Isolation of Flowing Mesenteric Lymph in Mice to Quantify In Vivo Kinetics of Dietary Lipid Absorption and Chylomicron Secretion

The present protocol describes a detailed surgical protocol for isolating flowing intestinal lymph in response to intraduodenal nutrient infusions in mice. This allows for the physiological determination of total intestinal lipid absorption and chylomicron synthesis and secretion in response to various experimental nutrients.

식이 지질 흡수 및 유미미크론 분비의 생체 내 동역학을 정량화하기 위한 마우스의 흐르는 장간막 림프의 분리

Intestinal lipoproteins, especially triglyceride-rich chylomicrons, are a major driver of metabolism, inflammation, and cardiovascular diseases. However, ...