עבודה זו נתמכה על ידי תרומות של קליפורד הגברת זקן לבן גרהם נתרמו לקרן מחקר ה-NIH (AR059838) ל CB, מ-NIH (AR052791, AR044387-ערה, &amp; NS063298) כדי LTT, ועל ידי במכללת ביילור לרפואה cytometry ו-Cell מיון Core במימון ה-NIH (NCRR S10RR024574, NIAID AI036211, P30CA125123 &amp; NCI). התוכן הוא באחריות הבלעדית של הכותבים ולא בהכרח מייצגים את הדעות הרשמיות של NIH.

1. תא מכתים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכינו את התאים. התאים יש להשתמש תוך ומתרחב (60-80 מפגש%). סרום לילה להרעיב את התאים, התאים צלחת אותם 0,100,000 דולר היטב בינוני 0.5 מ&quot;ל סרום ללא בצלחת 24-היטב ובמקום בחממה (37 ° C, 5% CO 2) דקות 30-2 שעות להתאושש מ trypsinization. </li><li style=";text-align:right;direction:rtl"> מערבבים את נוגדנים ראשוניים ומשניים. מערבבים (5 μL של נוגדנים נגד GLUT4 ראשוני עם 1 נוגדן μL משני נגד עז עוף IgG מצומדות כדי AlexaFluor 488) x מספר בארות. דגירה במשך 10 דקות בטמפרטורת החדר, בחושך. </li><li style=";text-align:right;direction:rtl"> הכינו מלאי עבודה 2X של אינסולין על ידי דילול אותו בינוני. </li><li style=";text-align:right;direction:rtl"> בדוק אם התאים יש דבק או לא. </li><li style=";text-align:right;direction:rtl"> לערבב בעדינות להוסיף 6 נוגדן μL ו 0.5 מ&quot;ל של המניה 2X שלך עובד של אינסולין כדי בארות המכיל את התאים. הימנע מכל מערבולת של המדיום. דגירה (37 מעלות צלזיוס, 5% CO 2) במשך 30 דקות, בחושך. </li><li style=";text-align:right;direction:rtl"> תקן את התאים על ידי הוספת 0.5 מ&quot;ל של PBS PFA + 1% זה טוב, בלי לטלטל את התאים. דגירה במשך 20 דקות בטמפרטורת החדר, בחושך. </li><li style=";text-align:right;direction:rtl"> אם התאים שלך יש דבק הבארות, בעדינות להרים אותם עם מרים תא. העברת התאים (1 סה&quot;כ מ&quot;ל) לתוך הצינורות להשתלב cytometer תזרים שלך צנטריפוגות כדי גלולה התאים. </li><li style=";text-align:right;direction:rtl"> שטפו את הכדור פעמיים עם 1 מ&quot;ל PBS ו resuspend ב 0.4 מ&quot;ל PBS + 1% PFA. בשלב זה, התאים יכולים להיות עטוף בנייר כסף ולאחסן במקרר (4-8 מעלות צלזיוס) או נלקח cytometer את זרימת לרכישת נתונים מיידית. </li></ol> 2. Data Acquisition <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> קביעת שער רחב סביב תאים חיים לפזר את הצד (SSC) מול פאנל קדימה (FCS) פיזור. השתמש צינור שלילי (תאים לא שטופלו באינסולין או תאים &quot;מוכתם&quot; עם פקד אלוטיפ) כדי להגדיר פרמטר FL1 שלך. ודא השיא שלך לחלוטין בלוח, לא לחתוך בצד התחתון. </li><li style=";text-align:right;direction:rtl"> רוכשת את נתוני לפחות 10,000 תאים לכל צינור. </li></ol> 3. ניתוח נתונים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ציור השער סביב תאים חיים לעומת SSC FSC פאנל (איור 1A). מגרש היסטוגרמות של עוצמת הקרינה לעומת 488 AlexaFluor מספר התאים בתוך השער לחיות התא (איור 1B). ניתן כיסוי היסטוגרמות שונות כדי להמחיש הבדלים אבל quantitation, לקבוע את עוצמת הקרינה מתכוון החציוני של האוכלוסייה בכל תא להשתמש במספרים אלה להשוואות שלך (תרשים 1C). </li><li style=";text-align:right;direction:rtl"> נרמל את הנתונים לערך המתקבל בהעדר אינסולין, כפי שמוצג 1C תרשימים 1D. </li></ol> 4. נציג תוצאות ב myoblasts מבודד מאדם בריא (ללא תנגודת לאינסולין), אינסולין משרה טרנסלוקציה מינון תלויי של GLUT4 מן הציטופלסמה אל הממברנה פלזמה (איור 1, משמאל). לעומת זאת, האינסולין אינו לגרום GLUT4 טרנסלוקציה על קרום התא של myoblasts מבודד חולה עם עמידות לאינסולין (איור 1, מימין). <img src="/files/ftp_upload/2429/2429fig1.jpg" alt="איור 1" /> באיור 1. דוגמה של ניסוי מכתים. Myoblasts בודדו מתורם ללא תנגודת לאינסולין (משמאל) מחולה עם עמידות לאינסולין (מימין). A, SSC (פיזור בצד) לעומת FCS (קדימה פיזור) העלילה עם שער סביב תאים חיים. B, תאים סגורה ב , מוכתם נוגדנים נגד epitope חיצוני של GLUT4, והשאיר unstimulated (אדום) או מגורה עם 1 ננומטר אינסולין (כחול), 10 ננומטר (כתום), או 100 ננומטר (ירוק). למען הבהירות דמות, אנחנו רק להציג נתונים בלי ועם אינסולין 100 ננומטר עבור התאים עם עמידות לאינסולין. C, ממוצע עוצמות הקרינה (MFI) של היסטוגרמות שמוצג B היו מנורמל ל MFI של תאים שמאל unstimulated (לא אינסולין). D, מגרשים של הנתונים מנורמל MFI מ היסטוגרמות המוצג ב

<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>

אין ניגודי אינטרסים הכריז.

אנחנו הוכיחו טכניקה מהירה לכימות טרנסלוקציה של GLUT4 מן הציטופלסמה אל קרום הפלזמה של תאים על ידי cytometry הזרימה. GLUT4 מתבטאת רק transiently על קרום התא של תאים הוא endocytosed. מאז נוגדנים מחויב להישאר צמודים GLUT4, אפילו במהלך אנדוציטוזה, האות הקרינה נותן את הסכום הכולל של GLUT4 כי נחשף הקרום פלזמה במהלך תקופת הדגירה של נבחר בנוכחות של אינסולין. טכניקה זו יכולה לחול גם על לימוד טרנסלוקציה של חלבונים אחרים בתא תאיים על ממברנת פלזמה, ובלבד נוגדן טוב להפנות epitope חיצוני של החלבון עניין זמין. לדוגמה, assay דומה כיום נפוץ כדי להעריך את שיעור degranulation ציטוטוקסיות של לימפוציטים מסוג T ו לימפוציטים הרוצח טבעי על ידי מדידת טרנסלוקציה של CD107a אל הממברנה הפלסמטית 4,5 שלהם. טרנסלוקציה של phosphatidylserine מהצד הפנימי של קרום התא אל הצד החיצוני במהלך האפופטוזיס התא או נמק יכול גם להתגלות על ידי cytometry זרימה, באמצעות fluorophore שכותרתו Annexin V 6. מלבד המהירות של assay, cytometry זרימה מציג את היתרון המאפשר לימוד של טרנסלוקציה חלבון קרום התא פלזמה באוכלוסיות מעורבות. אכן, אפשר לשלב מכתים עבור סמנים משנה התא לחלבון של עניין, ואחריו רכישת נתונים על cytometer רב לייזר זרימה 7. כמויות של נוגדנים שניתן בסעיף 1.2 של פרוטוקול הם נקודת התחלה: אנו ממליצים לכיל הנוגדנים שלך עם תאים העניין שלך כדי לקבוע את ריכוז הנוגדן המינימום שנותן לך אות מקסימלית. מגוון התחלה טובה הנוגדן אנטי אנושי ראשוני GLUT4 השתמשנו כאן יהיה μL 1-10 לכל שפופרת (כלומר 0.2-2 נוגדן מיקרוגרם לכל צינור), כל צינור המכיל לא יותר מ 1 מיליון תאים. עבור נוגדנים ראשוניים אחרים, עיין בהוראות של הספק. נורמליזציה של הנתונים כנדרש autofluorescence של תאים ישתנו מתורם לתורם ו מניסוי לניסוי.

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		<div>
		<table border="1">
		<thead>
		<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

הגלוקוז הוא המקור העיקרי של אנרגיה לגוף, הדורשים ויסות מתמיד של הריכוז בדם שלה. שחרור אינסולין על ידי הלבלב גורם ספיגת הגלוקוז על ידי רקמות רגישות לאינסולין, בעיקר את המוח, שרירי השלד, וכן adipocytes. חולים שסובלים מסוכרת מסוג 2 ו / או השמנת יתר לעיתים קרובות לפתח עמידות לאינסולין אינם מסוגלים לשלוט הומאוסטזיס הגלוקוז שלהם. תובנות חדשות על מנגנוני עמידות לאינסולין עשויים לספק אסטרטגיות טיפול חדש לסוכרת מסוג 2. משפחת שפע של מובילי הגלוקוז מורכב מחברי thirteen מופץ על רקמות שונות בגוף 1. טרנספורטר הגלוקוז סוג 4 (GLUT4) הוא טרנספורטר העיקריים מתווכת ספיגת הגלוקוז על ידי רקמות רגישות לאינסולין כמו שריר השלד. עם קשירה של אינסולין לקולטן שלו, שלפוחית ​​המכילה GLUT4 translocate מן הציטופלסמה אל קרום התא, וגורם ספיגת הגלוקוז. צמצום GLUT4 טרנסלוקציה היא אחת הסיבות תנגודת לאינסולין סוג 2 סוכרת 2,3. טרנסלוקציה של GLUT4 מן הציטופלסמה אל קרום התא ניתן דמיינו ידי immunocytochemistry, באמצעות fluorophore-מצומדות GLUT4 נוגדנים ספציפיים. כאן אנו מתארים טכניקה לכמת סך של טרנסלוקציה GLUT4 קרום הפלזמה של תאים במהלך משך הנבחר, באמצעות cytometry הזרימה. פרוטוקול זה הוא מהיר (פחות מ 4 שעות, כולל דגירה עם אינסולין) ומאפשר ניתוח של כמה כמו 3000 או תאים רבים ככל 1,000,000 תאים לכל תנאי בניסוי יחיד. היא מסתמכת על אנטי GLUT4 נוגדנים להפנות epitope חיצוני של המשלח אשר נקלטות על ידי אותה ברגע זה נחשף המדיום תאיים לאחר טרנסלוקציה על קרום התא.

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

פרוטוקול זה מתאר שיטה מהירה לכמת את טרנסלוקציה של GLUT4 מן הציטופלסמה אל קרום התא של תאים על ידי cytometry הזרימה.

מדידה כמותית של טרנסלוקציה GLUT4 על קרום התא על ידי זרימת cytometry

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 Views.  Baylor College of Medicine. פרוטוקול זה מתאר שיטה מהירה לכמת את טרנסלוקציה של GLUT4 מן הציטופלסמה אל קרום התא של תאים על ידי cytometry הזרימה.

Video: 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.

מדידת חלבון ממברנה פלזמה חליפין Endocytic ידי Biotinylation הפיך

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

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: immunoprecipitation זוהה על ידי זרימת cytometry

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.

Cytometry זרימה טיהור תאים עכבר meiotic

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.

מיפוי דיפוזיה מולקולרית בקרום פלזמה ידי מרובות יעד Tracing (MTT)

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.

אופטימיזציה שיטות דוגמנות מכתימה והפצת נשק לניטור חלוקת תא באמצעות צבעי מעקב סלולרי

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.

ניתוח בזמן אמת של תעבורת רטינול ידי הקולטן ממברנה של חלבון מחייב פלזמת רטינול

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.

Immunodetection של החלבונים בממברנה חיצוניים על ידי cytometry הזרימה של מיטוכונדריה מבודדת

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.

מדידת Endoreduplication על ידי Cytometry זרימה של פקעת מבודד Protoplasts

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.

מדידה רגיש של Mitophagy מאת Flow Cytometry באמצעות תלויי-pH הכתב פלורסנט mt-Keima

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.

מדידת תפוקה גבוהה Dictyostelium discoideum Macropinocytosis על ידי Cytometry זרימה

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