אנו מודים לתכנית IMAT מכון הלאומי לסרטן מNIH למימון עבודה זו [R33 CA174616-01A1].

1. תא יעד הכנה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לשמור (תאים למשל. ראג&#39;י לימפומה) תאי מטרה בתקשורת צמיחה (תרבות בינונית 1640 RPMI, עם 10% בסרום חום מומת שור העובר (FBS), 4nM L- גלוטמין, ו -500 IU / פניצילין מיליליטר / סטרפטומיצין) ב 37 o C ב5% CO 2 באינקובטור. </li><li style=";text-align:right;direction:rtl"> צנטריפוגה המדגם לגלולת תאי היעד בשפופרת 15 מיליליטר. זמן צנטריפוגה משתנה עם תאים מסוגים שונים; צנטריפוגות 3 דקות ב 468 XG לתאי ראג&#39;י. </li><li style=";text-align:right;direction:rtl"> לשאוב ראשון כל בועות נוצרו על פני השטח של supernatant, ולהסיר את כל supernatant. </li><li style=";text-align:right;direction:rtl"> להוסיף 10 מיליליטר של PBS על הצינור. Re- להשעות תאים על ידי pipetting את הפתרון ומטה מספר פעמים כדי לשבור את גוש התא. </li><li style=";text-align:right;direction:rtl"> צנטריפוגה מדגם 3 דקות ב 468 x גרם. </li><li style=";text-align:right;direction:rtl"> לשאוב כל בועות נוצרו על פני השטח של supernatant, ולהסיר את כל supernatant. </li></ol> 2. הכנת Microarrays הסלולרי <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> Oמצורף תא btain 96 ערכות צלחת גם או מערך תאים 8 שקופיות קאמריות גם אחד (ראה טבלה של חומרים / חומרים כימיים). </li><li style=";text-align:right;direction:rtl"> החל 1.2 μl של Activator למגיב DNA מהערכה. מכסה את הבקבוקון, להפוך אותו, ובעדינות לנער כדי לערבב את הפתרון. </li><li style=";text-align:right;direction:rtl"> אפשר הפתרון להגיב במשך 20 דקות ב RT. </li><li style=";text-align:right;direction:rtl"> ליישם את הפתרון המעורב לגלולת תאי היעד (בצינור 15 מיליליטר). </li><li style=";text-align:right;direction:rtl"> להוסיף צבעי cytosolic הקרינה ירוקים בריכוז סופי של 500 ננומטר. </li><li style=";text-align:right;direction:rtl"> דגירה תאי היעד עם הפתרון המעורב והצבע למשך 30 דקות ב RT על שייקר מסלולית. </li><li style=";text-align:right;direction:rtl"> לשטוף את התאים פעמיים עם 5 מיליליטר PBS, וresuspend תאי תקשורת 10 מיליליטר צמיחה. </li><li style=";text-align:right;direction:rtl"> החל מהפתרון 100 μl מצעד 2.7 היטב כל אחד. לספור את מספר התאים על ידי שימוש בhemacytometer. הפוך מספרים סלולריים בטוחים בטווח שבין 5 × 4-1 אוקטובר × 10 6 תאים לכל טוב. </li><li style=";text-align:right;direction:rtl"> לאחר 10-15 דקות של דגירה על RT, לשטוף בעדינות את הדגימות פעמיים עם 200-300 תקשורת צמיחת μl להסיר כל תאים מאוגד. הערה: על 5-10 תקשורת צמיחת μl נשארה בכל אחד גם לאחר כביסה. </li></ol> 3. דם מלא Cytotoxicity מבחני <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> להוסיף 180 μl של כל דם אנושי לכל דגימה היטב. </li><li style=";text-align:right;direction:rtl"> השג פתרון נוגדן Anti-CD20 בשעה 5 מ&quot;ג / מיליליטר, ולבצע דילול סדרתי לעשות 50, 20, 10, 5, 2, 1, 0.5 מיקרוגרם / מיליליטר של תמיסת נוגדן Anti-CD20 בצינורות 1.5 מיליליטר. הוסף 20 μl של כל ריכוז של נוגדני Anti-CD20 מדגם זה גם מה שהופך את triplicates. </li><li style=";text-align:right;direction:rtl"> להוסיף יודיד Propidium בריכוז סופי של 500 ננומטר היטב כל אחד. </li><li style=";text-align:right;direction:rtl"> דגירה את הצלחת וכתוצאה מכך ב 37 ° C עם 5% CO 2 במשך 16 שעות. </li><li style=";text-align:right;direction:rtl"> תמונת התאים בשקופיות או 96 צלחות גם עם מערכת הדמיה אוטומטית עם הגדלה 8X. </li><li style=";text-align:right;direction:rtl"> השתמש בתכנית ספירת תאים של מערכת ההדמיה האוטומטית לכמת את התוצאות. הערה: התוכנה של מערכת ההדמיה האוטומטית מספקת פשוטהצעד אחר צעד פעולות לספירת תאים. ניתן למצוא בפרוטוקול של שימוש בתוכנה זו באתר האינטרנט של הספק. </li><li style=";text-align:right;direction:rtl"> בחר [תוצאות סקירה] מהדף הראשי של התוכנה. </li><li style=";text-align:right;direction:rtl"> בחר [הקובץ שנשמר לדוגמות]. </li><li style=";text-align:right;direction:rtl"> השער הראשון FITC אומר עוצמה&gt; 50, אז שער TxRed אומרת עוצמה&gt; 20. </li><li style=";text-align:right;direction:rtl"> בחר [סקירת פלייט]. </li><li style=";text-align:right;direction:rtl"> בחר [שולחנות יצוא]. הערה: נוסחת הרג תא אחוז של התכנית מוצגת להלן: הרג% תא המטרה = (# של תאים המוכתמים שניהם ירוקים ואדום / # של תאים מוכתמים ירוק) * 100% </li></ol>

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יש סופרים לא אינטרסים כלכליים מתחרים.

אש&quot;ל הוא קריטי בכלי סינון אנטי-סרטני במבחנה עם רזולוציה תא בודדת 12-16, מנוצלת באופן אידיאלי לאחר הקרנות יעד מסורתיות כגון ה- CDC ומבחני ADCC 9-11, ולפני ניסויים בבעלי חיים פרה-קליניים. נכון לעכשיו, מבחני סינון המטרה העיקרית כגון CDC או מבחני ADCC כולם מבו?...

התקדמות שחלה באחרונה בתעשיית התרופות הובילה להתעניינות גוברת במימוש ההזדהויות הספציפיות של נוגדני תאים סרטניים וטיפולים בסרטן מותאמים אישית; עם זאת, כמה מכשולים נתקלו בתהליך. רק 5% מסוכנים שיש להם פעילות אנטי-סרטנית בפיתוח פרה-קליני ברישיון לאחר שהציג יעילות מספיקה בשלב הבדיקות השנייה-שלישית 1,2. האסטרטגיות פרה-קליני (הן במבחנה in vivo) הן הכי מוצלחות כדוגמאות רבות הראו כי תרופות אנטי-סרטניות מתנהגות באופן שונה באדם ובחיות מעבדה בעיקר בשל מרכיבי הדם השונים שלהם 3-5. כדי לתת מענה לצורך של פלטפורמת הקרנת סמים אנטי-סרטנית ולספק ביצוע צ&#39;ק-אין נקודה לפני ניסויים בבעלי חיים יקרים וניסויים קליניים, assay אדם שלם cytotoxicity הדם (אש&quot;ל) מוצע להערכת יעילות תרופה אנטי-סרטנית בסביבה ביולוגית רלוונטית יותר.assay cytotoxicity דם כל יכול לשמש כדי להעריך את התגובה של תאים בודדים לנוגדנים ותרופות פוטנציאליים אחרות בכל דם אנושי. אש&quot;ל הוא פותח על ידי שילוב טכנולוגיית מיקום תא תפוקה גבוהה עם תפוקה גבוהה וגבוה-תוכן הדמיה 6. על ידי ניצול מערכת הדמיה אוטומטית, מספר שני תאי החיים ומתים ניתן לקבוע ברמה גבוהה של דיוק. בשל העובדה שתאי המטרה הם משותקים באותו מישור המוקד, אש&quot;ל הוא מסוגל לספק ניתוח רעיל כמותי בזמן אמת, ללא ההסרה של תאי דם אדומים. יתר על כן, מערכת ההדמיה האוטומטית מספקת מספר יתרונות כגון שרק תאי היעד שהגיעו לקריטריונים שצוינו (לדוגמא., תאים שכותרתו fluorescently, ומורפולוגיה של תאים) הם מגודרים ומעובד. כמו כן, היא מאפשרת הייצור של 144 צלחות ביום. כתוצאה מכך, יכולת הדמיה זו ותפוקה מאפשרת ריצה של ההיי-גontent וניסויים תפוקה גבוהה בו זמנית. על ידי שילוב של אש&quot;ל ומערכת ההדמיה האוטומטית, ניתוח cytotoxicity תא כמותית גבוהה תפוקה ניתן להשיג בסביבה רלוונטית יותר ביולוגית.

<table border="0" cellpadding="0" cellspacing="0" width="761">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Name of Material/ Equipment</td>
			<td style="width:172px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:135px;">Name/Discription</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:336px;">Cell attachment 96 well plate kits&#160;</td>
			<td style="width:172px;">Adheren</td>
			<td style="width:119px;">AP9601</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:336px;">Suspension Single cell array 8 well chamber slide</td>
			<td style="width:172px;">Adheren</td>
			<td style="width:119px;">SS0801</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:336px;">Adherent Single cell array 8 well chamber slide</td>
			<td style="width:172px;">Adheren</td>
			<td style="width:119px;">SS0802</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Lymphoma cell line CD20+</td>
			<td style="width:172px;">ATCC</td>
			<td style="width:119px;">CCL-86</td>
			<td style="width:135px;">Raji cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Lymphoma cell line CD20-</td>
			<td style="width:172px;">ATCC</td>
			<td style="width:119px;">CRL-8083</td>
			<td style="width:135px;">MC/CAR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">RPMI 1640 with L-glutamine</td>
			<td style="width:172px;">Life Technologies</td>
			<td style="width:119px;">11875-119</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Fetal Bovine Serum</td>
			<td style="width:172px;">Thermo</td>
			<td style="width:119px;">SH30070.01HI</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Peni/Strep</td>
			<td style="width:172px;">Life Technologies</td>
			<td align="right" style="width:119px;">15070063</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Cytosolic dye</td>
			<td style="width:172px;">Life Technologies</td>
			<td style="width:119px;">C7025</td>
			<td style="width:135px;">Cell Tracker Green</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Rituxan (Biosimilar)&#160;</td>
			<td style="width:172px;">Eureka Therapeutics</td>
			<td style="width:119px;"></td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Human whole blood</td>
			<td style="width:172px;">Allcells</td>
			<td style="width:119px;">WB001</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Propidium Iodide</td>
			<td style="width:172px;">Sigma</td>
			<td style="width:119px;">P4170-10MG</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Automatic imaging system</td>
			<td style="width:172px;">Molecular Devices</td>
			<td style="width:119px;">Contact Vendor</td>
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real-time cytotoxicity assays in human whole blood

Assay חי המבוסס על תא כולו דם רעיל (אש&quot;ל), המאפשר גישה למידע זמני של cytotoxicity התא הכוללת הוא פותח עם טכנולוגיית מיקום תא תפוקה גבוהה. אוכלוסיות התאים סרטניים הממוקדות מתוכנות מראש ראשונה לחוסר מעש לתבנית מערך, ושכותרתו עם צבעי ניאון cytosolic ירוקים. לאחר הקמת מערך תאים, תרופות נוגדנים מתווספות בשילוב עם כל דם אנושי. יודיד Propidium (PI) הוא הוסיף אז להעריך מוות של תאים. מערך התאים מנותח עם מערכת הדמיה אוטומטית. בעוד צבע cytosolic תוויות אוכלוסיות תאים סרטניים הממוקדות, PI תוויות אוכלוסיות תאי גידול המתים. כך, שיעור הרג תאים סרטני היעד ניתן לכמת על ידי חישוב המספר של הישרדות בתאים ממוקדים למספר התאים הממוקדים מתים. בשיטה זו, חוקרים יכולים לגשת תלוי זמן ומידע cytotoxicity התא תלוי במינון. למרבה הפלא, אין רדיו מסוכןכימיקלים המשמשים. אש&quot;ל מוצג כאן נבדק עם לימפומה, לוקמיה, ושורות תאי גידולים מוצקות. לכן, אש&quot;ל מאפשר לחוקרים להעריך את יעילות תרופה במצב רלוונטי ביותר ex vivo.

אנטי-CD20 נוגדנים ותאים לימפומה (תאי ראג&#39;י וMC / CAR) נבחר כמודל למערכת כדי להדגים את כל assay cytotoxicity הדם (אש&quot;ל) 7,8. היו תאי ראג&#39;י מספר עותק גבוה של CD20 על פני התא, ואילו תאי MC / CAR מספר נמוך עותק של CD20 על הממברנה שלהם. תאים ממוקדים הוכתמו ראשון ירוקים עם צבעי cytosolic הקרינה...

Watch this Scientific Journal Video about Real-time Cytotoxicity Assays in Human Whole Blood at JoVE.com

Real-time Cytotoxicity Assays in Human Whole Blood

Assay כל cytotoxicity הדם (אש&quot;ל) הוא assay cytotoxicity שפותח על ידי שילוב טכנולוגיית מיקום תא תפוקה גבוהה עם מיקרוסקופ פלואורסצנטי ועיבוד תמונה אוטומטי. כאן, אנו מתארים כיצד ניתן לנתח תאי הלימפומה שטופלו בנוגדן anti-CD20 בזמן אמת בכל דם אדם כדי לספק ניתוח cytotoxicity סלולארי כמותי.

בזמן אמת Cytotoxicity מבחני בדם כל אדם

A live cell-based whole blood cytotoxicity assay (WCA) that allows access to temporal information of the overall cell cytotoxicity is developed with ...

real-time-cytotoxicity-assays-in-human-whole-blood

Research

JoVE Journal

Medicine

12.1K Views.  Adheren, Inc.  Assay כל cytotoxicity הדם (אש"ל) הוא assay cytotoxicity שפותח על ידי שילוב טכנולוגיית מיקום תא תפוקה גבוהה עם מיקרוסקופ פלואורסצנטי ועיבוד תמונה אוטומטי. כאן, אנו מתארים כיצד ניתן לנתח תאי הלימפומה שטופלו בנוגדן anti-CD20 בזמן אמת בכל דם אדם כדי לספק ניתוח cytoto...

Video: Real-time Cytotoxicity Assays in Human Whole Blood

Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle uptake in tumors at the microscopic level1,2,3, highlighting the utility of a suitable xenograft model in which to perform detailed uptake analyses. Here, we use high-resolution intravital imaging to evaluate nanoparticle uptake in human tumor xenografts in a modified, shell-less chicken embryo model. The chicken embryo model is particularly well-suited for these in vivo analyses because it supports the growth of human tumors, is relatively inexpensive and does not require anesthetization or surgery 4,5. Tumor cells form fully vascularized xenografts within 7 days when implanted into the chorioallantoic membrane (CAM) 6. The resulting tumors are visualized by non-invasive real-time, high-resolution imaging that can be maintained for up to 72 hours with little impact on either the host or tumor systems. Nanoparticles with a wide range of sizes and formulations administered distal to the tumor can be visualized and quantified as they flow through the bloodstream, extravasate from leaky tumor vasculature, and accumulate at the tumor site. We describe here the analysis of nanoparticles derived from Cowpea mosaic virus (CPMV) decorated with near-infrared fluorescent dyes and/or polyethylene glycol polymers (PEG) 7, 8, 9,10,11. Upon intravenous administration, these viral nanoparticles are rapidly internalized by endothelial cells, resulting in global labeling of the vasculature both outside and within the tumor7,12. PEGylation of the viral nanoparticles increases their plasma half-life, extends their time in the circulation, and ultimately enhances their accumulation in tumors via the enhanced permeability and retention (EPR) effect 7, 10,11. The rate and extent of accumulation of nanoparticles in a tumor is measured over time using image analysis software. This technique provides a method to both visualize and quantify nanoparticle dynamics in human tumors.

We present a novel approach to quantify nanoparticle localization in the vasculature of human xenografted tumors using dynamic, real-time intravital imaging in an avian embryo model.

הערכה של ספיגת nanoparticle ב גידולים בזמן אמת באמצעות הדמיה Intravital

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle ...

Rapid Point-of-Care Assay of Enoxaparin Anticoagulant Efficacy in Whole Blood

There is the need for a clinical assay to determine the extent to which a patient's blood is effectively anticoagulated by the low-molecular-weight-heparin (LMWH), enoxaparin. There are also urgent clinical situations where it would be important if this could be determined rapidly. The present assay is designed to accomplish this. We only assayed human blood samples that were spiked with known concentrations of enoxaparin. The essential feature of the present assay is the quantification of the efficacy of enoxaparin in a patient's blood sample by degrading it to complete inactivity with heparinase. Two blood samples were drawn into Vacutainer tubes (Becton-Dickenson; Franklin Lakes, NJ) that were spiked with enoxaparin; one sample was digested with heparinase for 5 min at 37 &deg;C, the other sample represented the patient's baseline anticoagulated status. The percent shortening of clotting time in the heparinase-treated sample, as compared to the baseline state, yielded the anticoagulant contribution of enoxaparin. We used the portable, battery operated Hemochron 801 apparatus for measurements of clotting times (International Technidyne Corp., Edison, NJ). The apparatus has 2 thermostatically controlled (37 &deg;C) assay tube wells. We conducted the assays in two types of assay cartridges that are available from the manufacturer of the instrument. One cartridge was modified to increase its sensitivity. We removed the kaolin from the FTK-ACT cartridge by extensive rinsing with distilled water, leaving only the glass surface of the tube, and perhaps the detection magnet, as activators. We called this our minimally activated assay (MAA). The use of a minimally activated assay has been studied by us and others. 2-4 The second cartridge that was studied was an activated partial thromboplastin time (aPTT) assay (A104). This was used as supplied from the manufacturer. The thermostated wells of the instrument were used for both the heparinase digestion and coagulation assays. The assay can be completed within 10 min. The MAA assay showed robust changes in clotting time after heparinase digestion of enoxaparin over a typical clinical concentration range. At 0.2 anti-Xa I.U. of enoxaparin per ml of blood sample, heparinase digestion caused an average decrease of 9.8% (20.4 sec) in clotting time; at 1.0 I.U. per ml of enoxaparin there was a 41.4% decrease (148.8 sec). This report only presents the experimental application of the assay; its value in a clinical setting must still be established.

Demonstration of a rapid quantitative assay of the inhibition of blood coagulation by the low-molecular-weight-heparin, enoxaparin. The contribution of enoxaparin is assessed by removing its influence through digestion with heparinase. A fuller description of the assay is detailed in our published paper.1 The assay still requires clinical confirmation.

Point-of-Care Assay המהיר של יעילות קרישת הדם enoxaparin בכל

There is the need for a clinical assay to determine the extent to which a patient's blood is effectively anticoagulated by the ...

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR (qPCR)

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process including development1, apoptosis2, and cell cycle regulation3. MiRNAs are estimated to regulate the expression of 30% to 90% of human genes4 by binding to their target messenger RNAs (mRNAs)5. Widespread dysregulation of miRNAs has been reported in various diseases and cancer subtypes6. Due to their prevalence and unique structure, these small molecules are likely to be the next generation of biomarkers, therapeutic agents and/or targets.
Methods used to investigate miRNA expression include SYBR green I dye- based as well as Taqman-probe based qPCR. If miRNAs are to be effectively used in the clinical setting, it is imperative that their detection in fresh and/or archived clinical samples be accurate, reproducible, and specific. qPCR has been widely used for validating expression of miRNAs in whole genome analyses such as microarray studies7. The samples used in this protocol were from patients who underwent radical prostatectomy for clinically localized prostate cancer; however other tissues and cell lines can be substituted in. Prostate specimens were snap-frozen in liquid nitrogen after resection. Clinical variables and follow-up information for each patient were collected for subsequent analysis8.
Quantification of miRNA levels in prostate tumor samples. The main steps in qPCR analysis of tumors are: Total RNA extraction, cDNA synthesis, and detection of qPCR products using miRNA-specific primers. Total RNA, which includes mRNA, miRNA, and other small RNAs were extracted from specimens using TRIzol reagent. Qiagen's miScript System was used to synthesize cDNA and perform qPCR (Figure 1). Endogenous miRNAs are not polyadenylated, therefore during the reverse transcription process, a poly(A) polymerase polyadenylates the miRNA. The miRNA is used as a template to synthesize cDNA using oligo-dT and Reverse Transcriptase. A universal tag sequence on the 5' end of oligo-dT primers facilitates the amplification of cDNA in the PCR step. PCR product amplification is detected by the level of fluorescence emitted by SYBR Green, a dye which intercalates into double stranded DNA. Specific miRNA primers, along with a Universal Primer that binds to the universal tag sequence will amplify specific miRNA sequences. 
The miScript Primer Assays are available for over a thousand human-specific miRNAs, and hundreds of murine-specific miRNAs. Relative quantification method was used here to quantify the expression of miRNAs. To correct for variability amongst different samples, expression levels of a target miRNA is normalized to the expression levels of a reference gene. The choice of a gene on which to normalize the expression of targets is critical in relative quantification method of analysis. Examples of reference genes typically used in this capacity are the small RNAs RNU6B, RNU44, and RNU48 as they are considered to be stably expressed across most samples. In this protocol, RNU6B is used as the reference gene.

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR qPCR

Quantitative Real Time polymerase chain reaction (qPCR) is a rapid and sensitive method to investigate the expression levels of various microRNA (miRNA) molecules in tumor samples. Using this method expression of hundreds of different miRNA molecules can be amplified, quantified, and analyzed from the same cDNA template.

MicroRNA איתור גידולים הערמונית ב כמותי בזמן אמת PCR (QPCR)

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process ...

Movement Retraining using Real-time Feedback of Performance

Any modification of movement - especially movement patterns that have been honed over a number of years - requires re-organization of the neuromuscular patterns responsible for governing the movement performance. This motor learning can be enhanced through a number of methods that are utilized in research and clinical settings alike. In general, verbal feedback of performance in real-time or knowledge of results following movement is commonly used clinically as a preliminary means of instilling motor learning. Depending on patient preference and learning style, visual feedback (e.g. through use of a mirror or different types of video) or proprioceptive guidance utilizing therapist touch, are used to supplement verbal instructions from the therapist. Indeed, a combination of these forms of feedback is commonplace in the clinical setting to facilitate motor learning and optimize outcomes.
Laboratory-based, quantitative motion analysis has been a mainstay in research settings to provide accurate and objective analysis of a variety of movements in healthy and injured populations. While the actual mechanisms of capturing the movements may differ, all current motion analysis systems rely on the ability to track the movement of body segments and joints and to use established equations of motion to quantify key movement patterns. Due to limitations in acquisition and processing speed, analysis and description of the movements has traditionally occurred offline after completion of a given testing session.
This paper will highlight a new supplement to standard motion analysis techniques that relies on the near instantaneous assessment and quantification of movement patterns and the display of specific movement characteristics to the patient during a movement analysis session. As a result, this novel technique can provide a new method of feedback delivery that has advantages over currently used feedback methods.

Retraining abnormal movement patterns following injury or disease is a key component of physical rehabilitation. Recent advances in technology have permitted accurate assessment of movement during a variety of tasks, with near instantaneous quantification of results. This provides new opportunities for modification of faulty movement patterns in real time.

תנועת סבה באמצעות משוב בזמן אמת של ביצועים

Any modification of movement - especially movement patterns that have been honed over a number of years - requires re-organization of the neuromuscular ...

Cerebrospinal Fluid MicroRNA Profiling Using Quantitative Real Time PCR

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their translational repression. Changes in miRNA expression have been shown to be involved in the development of all major complex diseases. Furthermore, recent findings showed that miRNAs can be secreted to the extracellular environment and enter the bloodstream and other body fluids where they can circulate with high stability. The function of such circulating miRNAs remains largely elusive, but systematic high throughput approaches, such as miRNA profiling arrays, have lead to the identification of miRNA signatures in several pathological conditions, including neurodegenerative disorders and several types of cancers. In this context, the identification of miRNA expression profile in the cerebrospinal fluid, as reported in our recent study, makes miRNAs attractive candidates for biomarker analysis.
There are several tools available for profiling microRNAs, such as microarrays, quantitative real-time PCR (qPCR), and deep sequencing. Here, we describe a sensitive method to profile microRNAs in cerebrospinal fluids by quantitative real-time PCR. We used the Exiqon microRNA ready-to-use PCR human panels I and II V2.R, which allows detection of 742 unique human microRNAs. We performed the arrays in triplicate runs and we processed and analyzed data using the GenEx Professional 5 software.
Using this protocol, we have successfully profiled microRNAs in various types of cell lines and primary cells, CSF, plasma, and formalin-fixed paraffin-embedded tissues.

We describe a protocol of real time PCR to profile microRNAs in the cerebrospinal fluid (CSF). With the exception of RNA extraction protocols, the procedure can be extended to RNA extracted from other body fluids, cultured cells, or tissue specimens.

נוזל עמוד שדרת MicroRNA פרופיל שימוש כמותי בזמן אמת PCR

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their ...

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an increased risk of vascular morbidity.&#160;Yet, albeit&#160;the mounting clinical evidence, there is a paucity of clear studies of vascular toxicity and therefore the etiology of a heterogeneous group of vascular/cardiovascular disorders remains to be elucidated. Moreover, the mechanism that may underlie vascular toxicity can completely differ from the principles of chemotherapy-induced cardiotoxicity, which is related to direct myocyte injury. We have established a real-time, in vivo molecular imaging platform&#160;to evaluate&#160;the potential&#160;acute&#160;vascular toxicity of anti-cancer therapies.
We have set up a platform of in vivo, high-resolution molecular imaging&#160;in mice,&#160;suitable for visualizing vasculature within confined organs and reference blood vessels within the same individuals whereas each individual serve as its own control. Blood vessel walls were impaired after doxorubicin administration, representing a unique mechanism of vascular toxicity that may be the early event in end-organ injury. Herein, the method of fibered confocal fluorescent microscopy (FCFM) based imaging is described, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

We herein describe the method of fibered confocal fluorescent microscopy (FCFM) based imaging, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

בעקבות כימותרפיה כלי דם רעילות - בזמן אמת<em&gt; In vivo</em&gt; הדמיה של ירידת ערך כלי

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an ...

Assessment of Ovarian Cancer Spheroid Attachment and Invasion of Mesothelial Cells in Real Time

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not accurately model the behaviors of cancer cells within a nonadherent environment, as cancer cells inherently aggregate into multicellular structures which contribute to the metastatic process by attaching to and invading the peritoneal lining to form secondary tumors. To model this important stage of ovarian cancer metastasis, multicellular aggregates, or spheroids, can be generated from established ovarian cancer cell lines maintained under nonadherent conditions. To mimic the peritoneal microenvironment encountered by tumor cells in vivo, a spheroid-mesothelial co-culture model was established in which preformed spheroids are plated on top of a human mesothelial cell monolayer, formed over an extracellular matrix barrier. Methods were then developed using a real-time cell analyzer to conduct quantitative real time measurements of the invasive capacity of different ovarian cancer cell lines grown as spheroids. This approach allows for the continuous measurement of invasion over long periods of time, which has several advantages over traditional endpoint assays and more laborious real time microscopy image analyses. In short, this method enables a rapid, determination of factors which regulate the interactions between ovarian cancer spheroid cells invading through mesothelial and matrix barriers over time.

Ovarian cancer cell invasion into the mesothelial lining of the peritoneum is a dynamic process over time. Utilizing a real time analyzer, the invasive capacity of ovarian cancer cells in a spheroid-mesothelial cell co-culture model can be quantified over prolonged time periods, providing insights into factors regulating the metastatic process.

הערכה של קובץ מצורף סרטן השחלות אליפטית ופלישה של תאי mesothelial בזמן אמת

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not ...

Real-time X-ray Imaging of Lung Fluid Volumes in Neonatal Mouse Lung

At birth, the lung undergoes a profound phenotypic switch from secretion to absorption, which allows for adaptation to breathing independently. Promoting and sustaining this phenotype is critically important in normal alveolar growth and gas exchange throughout life. Several in vitro studies have characterized the role of key regulatory proteins, signaling molecules, and steroid hormones that can influence the rate of lung fluid clearance. However, in vivo examinations must be performed to evaluate whether these regulatory factors play important physiological roles in regulating perinatal lung liquid absorption. As such, the utilization of real time X-ray imaging to determine perinatal lung fluid clearance, or pulmonary edema, represents a technological advancement in the field. Herein, we explain and illustrate an approach to assess the rate of alveolar lung fluid clearance and alveolar flooding in C57BL/6 mice at post natal day 10 using X-ray imaging and analysis. Successful implementation of this protocol requires prior approval from institutional animal care and use committees (IACUC), an in vivo small animal X-ray imaging system, and compatible molecular imaging software.

We present a protocol to assess the rate of alveolar fluid clearance or pulmonary edema in neonatal mouse lung using X-ray imaging technology.

הדמית רנטגן בזמן אמת של כרכי נוזל ריאות עובריות עכבר ריאות

At birth, the lung undergoes a profound phenotypic switch from secretion to absorption, which allows for adaptation to breathing independently. Promoting ...

Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time PCR

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated death 1. Common sites of metastatic spread include lung, lymph node, brain, and bone 2. Mechanisms that drive metastasis are intense areas of cancer research. Consequently, effective assays to measure metastatic burden in distant sites of metastasis are instrumental for cancer research. Evaluation of lung metastases in mammary tumor models is generally performed by gross qualitative observation of lung tissue following dissection. Quantitative methods of evaluating metastasis are currently limited to ex vivo and in vivo imaging based techniques that require user defined parameters. Many of these techniques are at the whole organism level rather than the cellular level 3-6. Although newer imaging methods utilizing multi-photon microscopy are able to evaluate metastasis at the cellular level 7, these highly elegant procedures are more suited to evaluating mechanisms of dissemination rather than quantitative assessment of metastatic burden. Here, a simple in vitro method to quantitatively assess metastasis is presented. Using quantitative Real-time PCR (QRT-PCR), tumor cell specific mRNA can be detected within the mouse lung tissue.

This protocol describes how to use quantitative Real-time PCR (QRT-PCR) to detect tumor cell specific mRNA representing metastasis within the mouse lung tissue.

הערכה של ריאות גרורה במודלי גידול עכבר החלב על ידי בזמן אמת כמותי PCR

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated ...

Uncontrolled Hemorrhagic Shock Modeled via Liver Laceration in Mice with Real Time Hemodynamic Monitoring

Uncontrolled hemorrhage is an important cause of preventable deaths among trauma patients. We have developed a murine model of uncontrolled hemorrhage via a liver laceration that results in consistent blood loss, hemodynamic alterations, and survival.
Mice undergo a standardized resection of the left-middle lobe of the liver. They are allowed to bleed without mechanical intervention. Hemostatic agents can be administered as pre-treatment or rescue therapy depending on the interest of the investigator. During the time of hemorrhage, real-time hemodynamic monitoring via a left femoral arterial line is performed. Mice are then sacrificed, blood loss is quantified, blood is collected for further analysis, and organs are harvested for analysis of injury. Experimental design is described to allow for simultaneous testing of multiple animals.
Liver hemorrhage as a model of uncontrolled hemorrhage exists in the literature, primarily in rat and porcine models. Some of these models utilize hemodynamic monitoring or quantify blood loss but lack consistency. The present model incorporates quantification of blood loss, real-time hemodynamic monitoring in a murine model that offers the advantage of using transgenic lines and a high-throughput mechanism to further investigate the pathophysiologic mechanisms in uncontrolled hemorrhage.

Uncontrolled hemorrhage, an important cause of mortality among trauma patients, can be modeled using a standard liver laceration in a murine model. This model results in consistent blood loss, survival, and allows for testing hemostatic agents. This article provides the step-by-step process to perform this valuable model.

הלם Hemorrhagic בלתי מבוקר מעוצב באמצעות laceration הכבד בעכברים עם ניטור ההודינמי בזמן אמת

Uncontrolled hemorrhage is an important cause of preventable deaths among trauma patients. We have developed a murine model of uncontrolled hemorrhage via ...