이 작품은 부인의 클리포드 엘더 화이트 그레이엄의 보조금에 의해 지원되었다 LTT하는 NIH (AR052791, AR044387 - 아라, &amp; NS063298)에서, CB 연구 기금 &amp; NIH (AR059838)를 둘러 싸인하고, 의학 Cytometry의 베일러 대학에서 NIH (NCRR S10RR024574, NIAID AI036211, &amp; NCI P30CA125123)에서 자금 코어 정렬과 셀. 내용은 전적으로 저자의 책임이며 반드시 NIH의 공식 견해를 대변하지 않습니다.

1. 세포 스테 이닝 <ol><li> 셀을 준비합니다. 아직 적극적으로 (- 80 % 코플루엔스 60) 성장하는 동안 세포를 사용해야합니다. 혈청은 인큐베이터에서 24 잘 플레이트와 장소에서 0.5 ML 혈청없는 중간에 하루 잘 당 0,100,000에 판 이들 세포를 세포를 굶어 (37 ° C, 5 % CO 2) 30 분 - 복구 ~ 2 시간 trypsinization에서. </li><li> 기본 및 보조 항체를 섞는다. 믹스 (AlexaFluor 488에 복합 1 μL 보조 닭고기 방지 염소 IgG 항체와 기본 방지 GLUT4 항체의 5 μL) 웰스의 X 숫자. 어둠 속에서, 실온에서 10 분 알을 품다. </li><li> 중간에 diluting에 의해 인슐린의 2X 작동 재고를 준비합니다. </li><li> 세포가 준수하거나하지 않은 경우 확인하십시오. </li><li> 부드럽게 세포를 포함하는 우물에 인슐린의 2X 작업 주식 6 μL 항체 믹스와 0.5 ML를 추가합니다. 매체의 난기류를 피하십시오. 알을 품다 (37 ° C 5 % CO 2) 30 분, 어둠 인치 </li><li> 세포를 떨지 않고, 각 잘하는 PBS + 1% PFA 0.5 ML를 추가하여 세포를 수정. 어둠 속에서, 실온에서 20 분 동안 품어. </li><li> 당신의 세포가 우물을 준수있다면, 부드럽게 세포 리프터 그들을 리프트. 귀하의 흐름 cytometer에 맞게 및 펠릿 세포 원심 튜브에 세포를 (1 ML 합계) 전송. </li><li> 0.4 ML PBS 1 ML PBS와 resuspend로 두 번 펠렛 + 1퍼센트 PFA 씻으십시오. 이 시점에서, 세포는 호일에 싸여 수 있으며 냉장고에 보관 (4-8 ° C) 또는 즉시 데이터 수집에 대한 흐름 cytometer로 이동합니다. </li></ol> 2. 데이터 수집 <ol><li> 사이드 분산형 (SSC) 대 앞으로 분산형 (FCS) 패널에서 살아있는 세포 주변 브로드 게이트를 설정합니다. 귀하의 FL1 매개 변수를 설정하는 부정적인 튜브 (세포가 인슐린이나 isotype 컨트롤과 함께 &quot;스테인드&quot;세포로 치료되지 않음) 사용합니다. 당신의 정상이 패널에서 완벽 하단 측면에 상처하지 않도록하십시오. </li><li> 튜브 당 최소 10,000 세포에서 데이터를 수집. </li></ol> 3. 데이터 분석 <ol><li> SSC VS FSC 패널 (그림 1A)에서 살아있는 세포 주위에 게이트를 그립니다. AlexaFluor 488 형광 강도를 비교 라이브 세포 게이트 내의 세포 (그림 1B)의 숫자의 histograms에 플롯. 당신은 차이를 시각화하기 위해 다른 histograms을 오버레이하지만 quantitation 위해 각 셀에 인구에 대한 의미와 평균 형광 강도를 결정하고 비교 (그림 1C) 이러한 숫자를 사용할 수 있습니다. </li><li> 같은 인물 1C 및 1D에 표시된 인슐린의 부재에서 얻어진 값, 데이터를 정상화. </li></ol> 4. 대표 결과 건강한 개인 (NO 인슐린 저항)에서 고립 myoblasts에서 인슐린은 플라즈마 막 (그림 1, 왼쪽)으로 세포질에서 GLUT4의 복용 종속 translocation을 유도. 반대로, 인슐린은 인슐린 저항 (그림 1, 오른쪽)와 함께 환자의 격리 myoblasts의 플라즈마 막에 GLUT4 translocation을 유발하지 않습니다. <img src="/files/ftp_upload/2429/2429fig1.jpg" alt="그림 1" /> 그림 1. 염색법 실험의 예. 에 Myoblasts가 인슐린 저항없이 기증자 (왼쪽)로부터 인슐린 저항 (오른쪽)와 함께 환자에서 고립되었다. 살아있는 세포 주위에 게이트가있는 A, SSC (사이드 분산형) 대 FCS (전진 분산형) 플롯. B, 세포는 게이 티드 A, GLUT4의 외부 에피토프에 대한 항체 물들일하고, unstimulated (적색) 또는 인슐린 1 NM (파란색), 10 NM (오렌지), 또는 100 nm의 (녹색)과 자극 떠났다. 그림의 지혜로움에 대해, 우리는 인슐린 저항과 전지 100 NM의 인슐린없이 데이터를 표시합니다. C는 B에 표시된 histograms의 형광 강도 (MFI) 세포의 MFI에 표준되었습니다 (NO 인슐린) unstimulated 왼쪽을 뜻합니다. D는 histograms에서 표준 MFI 데이터의 음모는 B.에 표시

<ol>
	<li>Zhao, F. Q., Keating, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+properties+and+genomics+of+glucose+transporters.">Functional properties and genomics of glucose transporters.</a> Curr. Genomics. 8, 113-128 (2007).</li><li>Karnieli, E., Armoni, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+regulation+of+the+insulin-responsive+glucose+transporter+GLUT4+gene:+from+physiology+to+pathology.">Transcriptional regulation of the insulin-responsive glucose transporter GLUT4 gene: from physiology to pathology.</a> Am. J. Endocrinol. Metab. 295, 38-45 (2008).</li><li>Graham, T. E., Kahn, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-specific+alterations+of+glucose+transport+and+molecular+mechanisms+of+intertissue+communication+in+obesity+and+type+2+diabetes.">Tissue-specific alterations of glucose transport and molecular mechanisms of intertissue communication in obesity and type 2 diabetes.</a> Horm. Metab. Res. 39, 717-721 (2007).</li><li>Alter, G., Malenfant, J. M., Altfeld, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD107a+as+a+functional+marker+for+the+identification+of+natural+killer+cell+activity.">CD107a as a functional marker for the identification of natural killer cell activity.</a> J. Immunol. Methods. 294, 15-22 (2004).</li><li>Vermes, I., Haanen, C., Steffens-Nakken, H., Reutellingsperger, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+assay+for+apoptosis.+Flow+cytometric+detection+of+phosphatidylserine+expression+on+early+apoptotic+cells+using+fluorescein+labeled+Annexin+V.">A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labeled Annexin V.</a> J. Immunol. Methods. 184, 39-51 (1995).</li><li>Lugli, E., Roederer, M., Cossarizza, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Data+analysis+in+flow+cytometry:+the+future+just+started.">Data analysis in flow cytometry: the future just started.</a> Cytometry A. 77, 705-713 (2010).</li></ol>

관심 없음 충돌 선언하지 않습니다.

우리는 유동세포계측법에 의해 세포의 플라즈마 막으로 세포질에서 GLUT4의 translocation을 quantifying에 대한 신속한 기술을 증명하고있다. GLUT4는 transiently 세포의 플라즈마 막에서 표현하고 endocytosed입니다. 바운드 항체도 endocytosis 동안 GLUT4에 연결된 상태 때문에, 형광 신호가 인슐린의 존재에 부화의 선택한 기간 동안 플라즈마 막에 노출되었다 GLUT4의 총 금액을 제공합니다. 이 기술은 또한, 플라즈마 막에 세포내 구획에서 다른 단백질의 translocation을 연구에 적용할 관심의 단백질의 외부 에피토프로 이동 좋은 항체 사용할 수 있습니다 제공될 수 있습니다. 예를 들어, 비슷한 분석은 이제 일반적으로 4,5 자신의 플라즈마 막에 CD107a의 translocation을 측정하여 세포 독성 T의 lymphocytes 자연 킬러 lymphocytes의 degranulation의 속도를 평가하는 데 사용됩니다. 세포 apoptosis 또는 괴사 동안 바깥쪽으로 플라즈마 막의 안쪽에서 phosphatidylserine의 translocation도 형광단 - 라벨 Annexin V 6을 사용, 유동세포계측법에 의해 감지하실 수 있습니다. 분석의 인텔리 게다가, 유동세포계측법 혼합 세포 집단에서 플라즈마 막에 단백질 translocation 연구를 허용 장점을 제공합니다. 사실, 하나는 세포 집합 표시 및 멀티 레이저 흐름 cytometer 7 데이터 수집 다음 ​​관심의 단백질에 대한 얼룩을 결합할 수 있습니다. 프로토콜의 단락 1.2 주어진 항체의 금액은 출발점되며 우리는 당신이 당신에게 최대 신호를 제공하는 최소 항체 농도를 결정하기 위해 관심의 세포와 항체를 적정하다하는 것이 좋습니다. 우리가 사용한 주요 안티 - 인간 GLUT4 항체에 대한 좋은 시작 범위 튜브 당 1-10 μL (튜브 당 즉 0.2-2 μg 항체), 더 이상 백만 이상의 세포를 포함하는 각각의 튜브 것입니다. 다른 차 항체 들어, 공급 업체의 지침을 참조하십시오. 세포의 autofluorescence은 기증자로부터 기증하고 실험에서 실험을 다양하므로 데이터의 정상화가 필요합니다.

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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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<tr>
<td>Anti-human GLUT4 antibodies</td>
<td>&nbsp;</td>
<td>Santa Cruz Biotechnologies</td>
<td>sc-1606</td>
<td>Against an external epitope of GLUT4</td>
</tr>
<tr>
<td>Chicken anti-goat IgG conjugated to AlexaFluor 488</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>A-21467</td>
<td>&nbsp;</td>
<tr>
<td>Recombinant human insulin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>I2643</td>
<td>&nbsp;</td>
<tr>
<td>Cell lifters</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>29942-118</td>
<td>Cut to fit the wells</td>
</tr>
<tr>
<td>5 ml tubes for FACS</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>60819-138</td>
<td>Fit all BD machines</td>
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quantitative measurement of glut4 translocation to the plasma membrane by flow cytometry

포도당은 혈액 농도의 지속적인 규제를 요구, 신체 에너지의 주요 원천입니다. 췌장에서 인슐린 릴리스는 인슐린에 민감한 조직, 특히 뇌, 골격근, 그리고 adipocytes에 의해 포도당 이해를 유도. 타입 2 당뇨병 및 / 또는 비만의 고통 환자는 종종 인슐린 저항을 개발하고 그들의 포도당 항상성를 제어하는​​ 수 없습니다. 인슐린 저항의 메커니즘에 새로운 통찰력은 유형 2 당뇨병에 대한 새로운 치료 전략을 제공할 수 있습니다. 포도당 전송기의 GLUT 가족은 신체 전반에 걸쳐 하나 다른 조직에 분산 열세 명의 회원으로 구성되어 있습니다. 포도당 수송 유형 4 (GLUT4)는 주요 전송기는 같은 골격근과 같은 인슐린에 민감한 조직에 의해 mediates 포도당 이해되는. 의 수용체에 인슐린의 결합되면, GLUT4는 포도당 이해를 유도, 플라즈마 막으로 세포질에서 이동시키다 포함 vesicles. 감소 GLUT4 translocation 형 - 2 당뇨병 2,3에서 인슐린 저항의 원인 중 하나입니다. 세포질에서 플라즈마 막에 GLUT4의 translocation이 형광단 - 복합 GLUT4 특정 항체를 사용, immunocytochemistry에 의해 시각하실 수 있습니다. 여기, 우리는 유동세포계측법를 사용하여 선택한 기간 동안 세포의 플라즈마 막에 GLUT4 translocation 총 금액을 수치 기법을 설명합니다. 이 프로토콜은 빠른 속도이다 (인슐린과 부화를 포함한 미만 4 시간)과 하나의 실험에서 3000 셀 또는 조건마다 많은 백만으로 세포 같이 몇 가지의 분석을 수 있습니다. 그것은 전송기의 외부 에피토프로 이동 방지 GLUT4 항체에 의존되는 즉시이 플라즈마 막에 translocation 후 세포 매체 노출로에 바인딩.

Watch this Scientific Journal Video about Quantitative Measurement of GLUT4 Translocation to the Plasma Membrane by Flow Cytometry at JoVE.com

Quantitative Measurement of GLUT4 Translocation to the Plasma Membrane by Flow Cytometry

이 프로토콜은 유동세포계측법에 의해 세포의 플라즈마 막으로 세포질에서 GLUT4의 translocation을 수치 빠른 방법을 설명합니다.

유동세포계측법에 의해 플라즈마 막에 GLUT4 Translocation의 양적 측정

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces ...

quantitative-measurement-glut4-translocation-to-plasma-membrane-flow

Research

JoVE Journal

Biology

26.3K 조회수.  Baylor College of Medicine. 이 프로토콜은 유동세포계측법에 의해 세포의 플라즈마 막으로 세포질에서 GLUT4의 translocation을 수치 빠른 방법을 설명합니다.

비디오: Quantitative Measurement of GLUT4 Translocation to the Plasma Membrane by Flow Cytometry

Measuring Plasma Membrane Protein Endocytic Rates by Reversible Biotinylation

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins is responsible for a variety of key cellular events, including nutrient delivery, cellular excitability, and chemical signaling.  Many plasma membrane proteins are dynamically regulated by endocytic trafficking, which modulates protein function by altering protein surface expression.  The mechanisms that facilitate protein endocytosis are complex and are not fully understood for many membrane proteins.  In order to fully understand the mechanisms that control the endocytic trafficking of a given protein, it is critical that the protein s endocytic rate be precisely measured.  For many receptors, direct endocytic rate measurements are frequently achieved utilizing labeled receptor ligands.  However, for many classes of membrane proteins, such as transporters, pumps and ion channels, there is no convenient ligand that can be used to measure the endocytic rate.  In the present report, we describe a reversible biotinylation method that we employ to measure the dopamine transporter (DAT) endocytic rate.  This method provides a straightforward approach to measuring internalization rates, and can be easily employed for trafficking studies of most membrane proteins.

Regulated endocytosis governs the cell surface expression levels of the majority of membrane proteins. Here we utilize reducible, membrane impermeant biotinylation reagents to measure the endocytic rate of the dopamine transporter (DAT), a polytopic membrane protein. The method facilitates a straightforward approach to measuring the endocytic rate of most plasma membrane proteins.

가역 Biotinylation에 의해 플라즈마 막 단백질 Endocytic 요금 측정

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins ...

연구

생물학

IP-FCM:  Immunoprecipitation Detected by Flow Cytometry

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic principle extends that of sandwich ELISA, wherein the captured primary analyte can be detected together with other molecules physically associated within multiprotein complexes. The procedure involves covalent coupling of polystyrene latex microbeads with immunoprecipitating monoclonal antibodies (mAb) specific for a protein of interest, incubating these beads with cell lysates, probing captured protein complexes with fluorochrome-conjugated probes, and analyzing bead-associated fluorescence by flow cytometry. IP-FCM is extremely sensitive, allows analysis of proteins in their native (non-denatured) state, and is amenable to either semi-quantitative or quantitative analysis. As additional advantages, IP-FCM requires no genetic engineering or specialized equipment, other than a flow cytometer, and it can be readily adapted for high-throughput applications.

The IP-FCM method is presented, which allows a sensitive, robust, biochemical assessment of native protein-protein interactions, without requiring genetic engineering or large sample sizes.

IP - FCM : 유동세포계측법에 의해 감지 Immunoprecipitation

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic ...

Flow Cytometry Purification of Mouse Meiotic Cells

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the unique differentiation programs that are manifest during meiosis. Two principal methods have been developed to purify, to varying degrees, various meiotic fractions from both adult and immature animals: elutriation or Staput (sedimentation) using BSA and/or percoll gradients. Both of these methods rely on cell size and density to separate meiotic cells1-5. Overall, except for few cell populations6, these protocols fail to yield sufficient purity of the numerous meiotic cell populations that are necessary for detailed molecular analyses. Moreover, with such methods usually one type of meiotic cells can be purified at a given time, which adds an extra level of complexity regarding the reproducibility and homogeneity when comparing meiotic cell samples.
Here, we describe a refined method that allows one to easily visualize, identify, and purify meiotic cells, from germ cells to round spermatids, using FACS combined with Hoechst 33342 staining7,8. This method provides an overall snapshot of the entire meiotic process and allows one to highly purify viable cells from most stage of meiosis. These purified cells can then be analyzed in detail for molecular changes that accompany progression through meiosis, for example changes in gene expression9,10and the dynamics of nucleosome occupancy at hotspots of meiotic recombination11.

An efficient method to obtain highly purified viable meiotic fractions from mouse testis is described, which combines a refined cell dissociation protocol with fluorescent activated cell sorting (FACS). This method takes advantage of differences in the DNA content and nuclear density of discrete meiotic fractions.

마우스 Meiotic 셀 유동세포계측법의 정제

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the ...

Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing (MTT)

Our goal is to obtain a comprehensive description of molecular processes occurring at cellular membranes in different biological functions. We aim at characterizing the complex organization and dynamics of the plasma membrane at single-molecule level, by developing analytic tools dedicated to Single-Particle Tracking (SPT) at high density: Multiple-Target Tracing (MTT)1. Single-molecule videomicroscopy, offering millisecond and nanometric resolution1-11, allows a detailed representation of membrane organization12-14 by accurately mapping descriptors such as cell receptors localization, mobility, confinement or interactions.
We revisited SPT, both experimentally and algorithmically. Experimental aspects included optimizing setup and cell labeling, with a particular emphasis on reaching the highest possible labeling density, in order to provide a dynamic snapshot of molecular dynamics as it occurs within the membrane. Algorithmic issues concerned each step used for rebuilding trajectories: peaks detection, estimation and reconnection, addressed by specific tools from image analysis15,16. Implementing deflation after detection allows rescuing peaks initially hidden by neighboring, stronger peaks. Of note, improving detection directly impacts reconnection, by reducing gaps within trajectories. Performances have been evaluated using Monte-Carlo simulations for various labeling density and noise values, which typically represent the two major limitations for parallel measurements at high spatiotemporal resolution.
The nanometric accuracy17 obtained for single molecules, using either successive on/off photoswitching or non-linear optics, can deliver exhaustive observations. This is the basis of nanoscopy methods17 such as STORM18, PALM19,20, RESOLFT21 or STED22,23, which may often require imaging fixed samples. The central task is the detection and estimation of diffraction-limited peaks emanating from single-molecules. Hence, providing adequate assumptions such as handling a constant positional accuracy instead of Brownian motion, MTT is straightforwardly suited for nanoscopic analyses. Furthermore, MTT can fundamentally be used at any scale: not only for molecules, but also for cells or animals, for instance. Hence, MTT is a powerful tracking algorithm that finds applications at molecular and cellular scales.

Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing MTT

Multiple-Target Tracing is a homemade algorithm developed for tracking individually labeled molecules within the plasma membrane of living cells. Efficiently detecting, estimating and tracing molecules over time at high-density provide a user-friendly, comprehensive tool to investigate nanoscale membrane dynamics.

다중 타겟 추적 (MTT)에 의해 플라즈마 막의 분자 확산을 매핑

Our goal is to obtain a comprehensive description of molecular processes occurring at cellular membranes in different biological functions. We aim at ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

셀 추적 염료를 사용하여 전지 부문 모니터링에 최적화 된 착색 및 확산 모델링 방법

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

Real-time Analyses of Retinol Transport by the Membrane Receptor of Plasma Retinol Binding Protein

Vitamin A is essential for vision and the growth/differentiation of almost all human organs. Plasma retinol binding protein (RBP) is the principle and specific carrier of vitamin A in the blood. Here we describe an optimized technique to produce and purify holo-RBP and two real-time monitoring techniques to study the transport of vitamin A by the high-affinity RBP receptor STRA6. The first technique makes it possible to produce a large quantity of high quality holo-RBP (100%-loaded with retinol) for vitamin A transport assays. High quality RBP is essential for functional assays because misfolded RBP releases vitamin A readily and bacterial contamination in RBP preparation can cause artifacts. Real-time monitoring techniques like electrophysiology have made critical contributions to the studies of membrane transport. The RBP receptor-mediated retinol transport has not been analyzed in real time until recently. The second technique described here is the real-time analysis of STRA6-catalyzed retinol release or loading. The third technique is real-time analysis of STRA6-catalyzed retinol transport from holo-RBP to cellular retinol binding protein I (CRBP-I). These techniques provide high sensitivity and resolution in revealing RBP receptor's vitamin A uptake mechanism.

Here we describe an optimized technique to produce high-quality vitamin A/RBP complex and two real-time monitoring techniques to study vitamin A transport by STRA6, the RBP receptor.

플라즈마 레티놀 바인딩 단백질의 막 수용체의 레티놀 전송의 실시간 분석

Vitamin A is essential for vision and the growth/differentiation of almost all human organs.  Plasma retinol binding protein (RBP) is the principle and ...

Immunodetection of Outer Membrane Proteins by Flow Cytometry of Isolated Mitochondria

Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and pathophysiology. This protocol describes a technique where mitochondria isolated from rodent tissue are immunolabeled and analyzed by flow cytometry. Mitochondria are isolated from rodent spinal cords and subjected to a rapid enrichment step so as to remove myelin, a major contaminant of mitochondrial fractions prepared from nervous tissue. Isolated mitochondria are then labeled with an antibody of choice and a fluorescently conjugated secondary antibody. Analysis by flow cytometry verifies the relative purity of mitochondrial preparations by staining with a mitochondrial specific dye, followed by detection and quantification of immunolabeled protein. This technique is rapid, quantifiable and high-throughput, allowing for the analysis of hundreds of thousands of mitochondria per sample. It is applicable to assess novel proteins at the mitochondrial surface under normal physiological conditions as well as the proteins that may become mislocalized to this organelle during pathology. Importantly, this method can be coupled to fluorescent indicator dyes to report on certain activities of mitochondrial subpopulations and is feasible for mitochondria from the central nervous system (brain and spinal cord) as well as liver.

Described here is a method to detect and quantify mitochondrial outer membrane proteins by immunolabeling of mitochondria isolated from rodent tissue and analysis by flow cytometry. This method can be extended to assess functional aspects of mitochondrial subpopulations.

고립 된 미토콘드리아의 유동 세포 계측법에 의해 외부 막 단백질의 면역

Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and ...

Measuring Endoreduplication by Flow Cytometry of Isolated Tuber Protoplasts

Endoreduplication, the replication of a cell's nuclear genome without subsequent cytokinesis, yields cells with increased DNA content and is associated with specialization, development and increase in cellular size. In plants, endoreduplication seems to facilitate the growth and expansion of certain tissues and organs. Among them is the tuber of potato (Solanum tuberosum), which undergoes considerable cellular expansion in fulfilling its function of carbohydrate storage. Thus, endoreduplication may play an important role in how tubers are able to accommodate this abundance of carbon. However, the cellular debris resulting from crude nuclear isolation methods of tubers, methods that can be used effectively with leaves, precludes the estimation of the tuber endoreduplication index (EI). This article presents a technique for assessing tuber endoreduplication through the isolation of protoplasts while demonstrating representative results obtained from different genotypes and compartmentalized tuber tissues. The major limitations of the protocol are the time and reagent costs required for sample preparation as well as relatively short lifespan of samples after lysis of protoplasts. While the protocol is sensitive to technical variation, it represents an improvement over traditional methods of nuclear isolation from these large specialized cells. Possibilities for improvements to the protocol such as recycling enzyme, the use of fixatives, and other alterations are proposed.

The protocol described herein is a method for measuring endoreduplication within tubers of potato (Solanum tuberosum). It includes plasmolysis and protoplast extraction steps to decrease the noise and debris in downstream flow cytometric analysis.

격리 된 괴 경 Protoplasts의 Cytometry에 의해 측정 Endoreduplication

Endoreduplication, the replication of a cell's nuclear genome without subsequent cytokinesis, yields cells with increased DNA content and is associated ...

Sensitive Measurement of Mitophagy by Flow Cytometry Using the pH-dependent Fluorescent Reporter mt-Keima

Mitophagy is a process of selective removal of damaged or unnecessary mitochondria using autophagy machinery. Close links have been found between defective mitophagy and various human diseases, including neurodegenerative diseases, cancer, and metabolic diseases. In addition, recent studies have shown that mitophagy is involved in normal cellular processes, such as differentiation and development. However, the precise role of and molecular mechanisms underlying mitophagy require further study. Therefore, it is critical to develop a robust and convenient method for measuring changes in mitophagy activity. Here, we describe a detailed protocol for quantitatively assessing mitophagy activity through flow cytometry using the mitochondria-targeted fluorescent protein Keima (mt-Keima). This flow cytometry assay can analyze mitophagy activity more rapidly and sensitively than conventional microscopy- or immunoblotting-based methods. This protocol can be applied to analyze mitophagy activity in various cell types.

Mitophagy, the selective degradation of mitochondria, has been implicated in mitochondrial homeostasis and is deregulated in various human diseases. However, convenient experimental methods for measuring mitophagy activity are very limited. Here, we provide a sensitive assay for measuring mitophagy activity using flow cytometry.

형광 기자 흐름 Cytometry 사용 pH-종속 mt-Keima에 의해 Mitophagy의 중요 한 측정

Mitophagy is a process of selective removal of damaged or unnecessary mitochondria using autophagy machinery. Close links have been found between ...

High-throughput Measurement of Dictyostelium discoideum Macropinocytosis by Flow Cytometry

Large-scale non-specific fluid uptake by macropinocytosis is important for the proliferation of certain cancer cells, antigen sampling, host cell invasion and the spread of neurodegenerative diseases. The commonly used laboratory strains of the amoeba Dictyostelium discoideum have extremely high fluid uptake rates when grown in nutrient medium, over 90% of which is due to macropinocytosis. In addition, many of the known core components of mammalian macropinocytosis are also present, making it an excellent model system for studying macropinocytosis. Here, the standard technique to measure internalized fluid using fluorescent dextran as a label is adapted to a 96-well plate format, with the samples analyzed by flow cytometry using a high-throughput sampling (HTS) attachment.
Cells are fed non-quenchable fluorescent dextran for a pre-determined length of time, washed by immersion in ice-cold buffer and detached using 5 mM sodium azide, which also stops exocytosis. Cells in each well are then analyzed by flow cytometry. The method can also be adapted to measure membrane uptake and phagocytosis of fluorescent beads or bacteria.
This method was designed to allow measurement of fluid uptake by Dictyostelium in a high-throughput, labor and resource efficient manner. It allows simultaneous comparison of multiple strains (e.g. knockout mutants of a gene) and conditions (e.g. cells in different media or treated with different concentrations of inhibitor) in parallel and simplifies time-courses.

High-throughput Measurement of Dictyostelium discoideum Macropinocytosis by Flow Cytometry

Macropinocytosis, large-scale non-specific fluid uptake, is important in many areas of clinical biology including immunology, infection, cancer, and neurodegenerative diseases. Here, existing techniques have been adapted to allow high-throughput, single-cell resolution measurement of macropinocytosis in the macropinocytosis model organism Dictyostelium discoideum using flow cytometry.

Flow Cytometry 여 Dictyostelium discoideum Macropinocytosis의 높은 처리량 측정

Large-scale non-specific fluid uptake by macropinocytosis is important for the proliferation of certain cancer cells, antigen sampling, host cell invasion ...