This work is supported by the Joseph Alexander Foundation, the ASBMB Undergraduate Research Award, 2013-2014, and a Science Award Grant, Marymount Manhattan College, 2012-2013.

<ol>
	<li>Hammamieh, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Students+investigating+the+antiproliferative+effects+of+synthesized+drugs+on+mouse+mammary+tumor+cells.">Students investigating the antiproliferative effects of synthesized drugs on mouse mammary tumor cells.</a> Cell Biol Educ. 4 (3), 221-234 (2005).</li><li>Sykes, J. A., Whitescarver, J., Briggs, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Observations+on+a+cell+line+producing+mammary+tumor+virus.">Observations on a cell line producing mammary tumor virus.</a> J Natl Cancer Inst. 41 (6), 1315-1327 (1968).</li><li>Palombi, P. S. J., Snell, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Learning+about+Cells+as+Dynamic+Entities:+An+Inquiry-Driven+Cell+Culture+Project.">Learning about Cells as Dynamic Entities: An Inquiry-Driven Cell Culture Project.</a> Bioscene: Journal of College Biology Teaching. 33, 27-33 (2008).</li><li>Ledbetter, M. L. S., Lippert, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucose+Transport+in+Cultured+Animal+Cells:+An+Exercise+for+the+Undergraduate+Cell+Biology+Laboratory.">Glucose Transport in Cultured Animal Cells: An Exercise for the Undergraduate Cell Biology Laboratory.</a> Cell Biology Education. 1 (3), 76-86 (2002).</li><li>Weaver, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+Cells+Beating+in+Culture:+A+Laboratory+Exercise.">Cardiac Cells Beating in Culture: A Laboratory Exercise.</a> American Biology Teacher. 69, 407-410 (2007).</li><li>Marion, R. E., Gardner, G. E., Parks, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiweek+cell+culture+project+for+use+in+upper-level+biology+laboratories.">Multiweek cell culture project for use in upper-level biology laboratories.</a> Advances in Physiology Education. 36, 154-157 (2012).</li><li>Mozdziak, P. E. P., James, N., Carson, S. u. s. a. n. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Introductory+Undergraduate+Course+Covering+Animal+Cell+Culture+Techniques.">An Introductory Undergraduate Course Covering Animal Cell Culture Techniques.</a> Biochemistry and Molecular Biology Education. 32 (5), 319-322 (2004).</li><li>Anderson, L. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> A taxonomy for learning, teaching and assessing: A revision of Bloom's Taxonomy of educational objectives (Complete Edition). , (2001).</li><li>Centers for Disease Contol and Prevention. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Appendix A - Primary Containment for Biohazards: Selection, Installation and Use of Biological Safety Cabinets. , (2014).</li><li>Davis, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Basic Cell Culture: A Practical Approach. , (2002).</li><li>Algra, A. M., Rothwell, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+regular+aspirin+on+long-term+cancer+incidence+and+metastasis:+a+systematic+comparison+of+evidence+from+observational+studies+versus+randomised+trials.">Effects of regular aspirin on long-term cancer incidence and metastasis: a systematic comparison of evidence from observational studies versus randomised trials.</a> Lancet Oncol. 13 (5), 518-527 (2012).</li><li>Anand, P., Sundaram, C., Jhurani, S., Kunnumakkara, A. B., Aggarwal, 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=Curcumin+and+cancer:+an+'old-age'+disease+with+an+'age-old'+solution.">Curcumin and cancer: an 'old-age' disease with an 'age-old' solution.</a> Cancer Lett. 267 (1), 133-164 (2008).</li><li>Ararat, E., Sahin, I., Altundag, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+behind+the+aspirin+use+and+decreased+breast+cancer+incidence.">Mechanisms behind the aspirin use and decreased breast cancer incidence.</a> J BUON. 16 (1), 180 (2011).</li><li>Ararat, E., Sahin, I., Altundag, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aspirin+intake+may+prevent+metastasis+in+patients+with+triple-negative+breast+cancer.">Aspirin intake may prevent metastasis in patients with triple-negative breast cancer.</a> Med Oncol. 28 (4), 1308-1310 (2011).</li><li>Blandino, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metformin+elicits+anticancer+effects+through+the+sequential+modulation+of+DICER+and+c-MYC.">Metformin elicits anticancer effects through the sequential modulation of DICER and c-MYC.</a> Nat Commun. 3, 865 (2012).</li><li>Kunnumakkara, A. B., Anand, P., Aggarwal, 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=Curcumin+inhibits+proliferation,+invasion,+angiogenesis+and+metastasis+of+different+cancers+through+interaction+with+multiple+cell+signaling+proteins.">Curcumin inhibits proliferation, invasion, angiogenesis and metastasis of different cancers through interaction with multiple cell signaling proteins.</a> Cancer Lett. 269 (2), 199-225 (2008).</li><li>Burstein, D. E., Blumberg, P. M., Greene, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nerve+growth+factor-induced+neuronal+differentiation+of+PC12+pheochromocytoma+cells:+lack+of+inhibition+by+a+tumor+promoter.">Nerve growth factor-induced neuronal differentiation of PC12 pheochromocytoma cells: lack of inhibition by a tumor promoter.</a> Brain Res. 247 (1), 115-119 (1982).</li><li>Nazarali, S. A., Narod, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tamoxifen+for+women+at+high+risk+of+breast+cancer.">Tamoxifen for women at high risk of breast cancer.</a> Breast Cancer (Dove Med Press). 6, 29-36 (2014).</li><li>Cui, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-talk+between+HER2+and+MED1+regulates+tamoxifen+resistance+of+human+breast+cancer+cells).">Cross-talk between HER2 and MED1 regulates tamoxifen resistance of human breast cancer cells).</a> Cancer Res. 72 (21), 5625-5634 (2012).</li><li>Komm, B. S., Mirkin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+overview+of+current+and+emerging+SERMs.">An overview of current and emerging SERMs.</a> J Steroid Biochem Mol Biol. 143C, 207-222 (2014).</li><li>Kaplan, R. M., Satterfield, J. M., Kington, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Building+a+better+physician--the+case+for+the+new+MCAT.">Building a better physician--the case for the new MCAT.</a> N Engl J Med. 366 (14), 1265-1268 (2012).</li></ol>

המחברים לא קיבלו תמיכה כספית או דומה מMOTIC.

מודול מעבדה מוצג שמטרתו ללמד מגוון של נושאים בביולוגיה של תא בטכניקות המתקדמות של תרבית תאים של בעלי החיים. מודול משיג זאת על ידי ניתוח ההשפעות של מספר הכימיקלים אנטי-שגשוג על השכפול של תאים שמודל סרטן השד אנושי. Assay העיקרי מסתמך על הטכניקה הבסיסית של ספירת תאים ומציג...

לעתים קרובות בקורסי ביולוגיה כללית לתואר ראשון, בנושאים של רגולציה מחזור התא וסרטן נגעו אך לא נחקרו בפירוט כי רוחבו של תוכן בקורסים אלה משאיר מעט מאוד זמן לעומק. בנוסף, סטודנטים לתואר ראשון בביולוגיה אינם חשופים בדרך כלל לטכניקות המתקדמות הקשורות לתרבות תא של בעלי החיים. כדי לסייע לתלמידים לפתח הבנה עמוקה יותר של מושגים אלה, תוך יישום והניתוח מה שהם למדו, פעילות מעבדה פותחה כשינוי של וולטר ריד צבא מכון המחקר (WRAIR) הרחיב את פעילות המעבדה 1. מודול המעבדה משתמש ב, אסטרטגיה ניסיונית צעד חכם שכוללת גידול ואפיון מודל של תאי סרטן, בפיתוח ובביצוע שיטות ספירת תאים, ההקמה כמובן זמן אופטימלי ומינונים לטיפול בתאים עם סוכנים אנטי-שגשוג, וזיהוי מסלולי איתות תאים חריגים . הניסוי גם מאפשר פתוחהודעה -ended. רוב הטכניקות הנדרשות לפעילות זו יכולה להתבצע במעבדה הוראת ביולוגיה-טיפוסית. הפעילות מתחילה עם תלמידים המאפיינים את שיעור המורפולוגיה וצמיחה של קו התאים סרטני החלב עכבר (MMT), מודל לסרטן השד אנושי 2. סרטן השד נבחר כמודל הסרטן בגלל שכיחותה באוכלוסייה, היכרותה לתלמידים בגילי קולג &#39;, ונתונים נרחבים הזמינים. שורת תאי MMT נבחרה במיוחד משום שהוא בר השגה בקלות, מאופיין היטב, יש זמן הכפלה קצר וקל לגדול. בנוסף, תאי MMT הם תלויים באסטרוגן אשר עולה בקנה אחד עם רוב מקרי סרטן השד נשיים. תלמידים לאחר מכן לזהות מסלולי איתות תאים חריגים בתאי MMT על ידי טיפול בתאים עם תרופות כימותרפיות מנגנון פעולה שלו הוא גם established.The ריכוז של התרופות והאורך של הטיפולים מגוונים המאפשרים לתלמידיםכדי להעריך את ההשפעה של המשתנים הללו על קצב חלוקת תאים. Assay מפתח לפעילות זו הוא קביעת כדאיות תא, שפשוט דורשת ספירת תאים, באמצעות אחת משתי שיטות. כל שיטה תלויה בכישורי מיקרוסקופ חזקים. תלמידים לקבוע כדאיות תא על ידי שימוש סטנדרטי, שיטת hemocytometer ושיטת photomicroscopy רומן ולהציע. בהתבסס על הממצאים שלהם, הם יכולים להציע ולבחון שינויים בפעילות. תלמידים אז לייצג את הנתונים שלהם ולפרש את התוצאות כדי לחדד את השערתם ולתכנן אסטרטגיות ניסיוניות חדשות. פעילות מעבדה זה מתאים לתלמידי כיתה ט &#39;או בכיתה י&#39; ברמת התמחות במדעים הביולוגיים. הוא מרוכז במודול מעבדה של שבוע שניתן להשלים בשנה ראשונה, ביולוגיה כללית או שנה שנייה, כמובן ביולוגיה תאית / מולקולריים. כישורים דרושים להשלמה נכונה של הפעילות כוללים חשבון בסיסי ואלגברה, היכרות עם מערך של גמיומנויות מעבדה עפרות (למשל, pipetting, פתרון קבלת, טכניקה סטרילית), ניתוח נתונים, ניהול מיקרוסקופ האור והזמן בסיסי, יחד עם ידע מורה של תרבית תאים ותוכנת גיליון אלקטרוני. ריאגנטים הנדרשים כוללים מודל שורת התאים של בעלי חיים לסרטן (לדוגמא, תאי החלב עכבר גידול, MMT 2), סוכני כימותרפיה (למשל, טמוקסיפן, כורכום, מטפורמין, ואספירין), תקשורת והתרבות כחולה ותא trypan (למשל, בינוני חיונית המינימלית של הנשר ; EMEM) עם תוספי מזון מתאימים (למשל, סוס תורם וסרום שור עוברי). מכשירים הדרושים כוללים מיקרוסקופ הפוך אור עם קובץ מצורף מצלמה דיגיטלית, מחשב, 100 מ&quot;מ ו -24 צלחות תרבית רקמה היטב, CO 2 באינקובטור (או שווה ערך), ארון בטיחות ביולוגית (BSC; Class II), hemocytometer, ותוכנת מיקרוסקופיה דיגיטלית. ישנן דוגמאות טובות של פעילויות מעבדה ספציפיות המסתמכות על תרבות תאים של בעלי החיים לTEACסטודנטים לתואר ראשון h על מושגים בביולוגיה של תא 3. עם זאת רבים דורשים אספקה ​​או טכניקות שאינן נגישים בקלות (למשל, איזוטופים רדיואקטיביים, רקמת חיה של בעלי חיים, ציוד הדמיה מתקדם 1,4,5), מתארים פרוטוקולים שהם די מתקדמים (למשל, מתאימים לקורס 400 רמה 6), או דורשים פרויקטים ארוכים רב-שבוע סמסטר או 6,7. פעילות המעבדה המתוארת כאן היא פשוטה ויכולה להיות שנערכה בשבוע עם ציוד מעבדה משותף אחת. לסיכום, מעבדה מודול זה ביעילות מציג או מחזק את המושגים של מחזור תא, מסלולי איתות תאיים וסרטן תוך הקניית מיומנויות בסיסיות ומתקדמות במעבדה, ניתוח נתוני ניסוי, השיטה של ​​תרבית תאים של בעלי חיים ואת התהליך המדעי. מודול המעבדה הוא פשוט ונגיש מבחינה כלכלית והן מספק גמישות והזדמנות לחקירה פתוחה. הפעילות מעודדת את יצירתיות תלמידעל ידי מתן אסטרטגיה ניסיונית תבנית שפועלת כמדריך, אבל לא מתכון. והכי חשוב, מספק פעילות בכל תחומי הלימוד של טקסונומיה פורח 8 כפי שהוא דורש לזכור, הבנה, יישום, ניתוח, הערכה ויצירה על ידי העוסק תלמידים בתהליך שמושך אותם אל מחוץ לספר הלימוד ולתוך העולם של מחקר מדעי.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Tissue Culture Hood</td>
			<td>ESCO Labculture Reliant</td>
			<td></td>
			<td>Class II Type A2 Biological Safety Cabinet</td>
		</tr>
		<tr>
			<td>Waterjactor CO2 Incubator</td>
			<td>CEDCO</td>
			<td>Model 1510</td>
			<td></td>
		</tr>
		<tr>
			<td>Bright-line Hemocytometer</td>
			<td>American Optical</td>
			<td></td>
			<td>with two separate grids</td>
		</tr>
		<tr>
			<td>Motic Images Plus</td>
			<td></td>
			<td></td>
			<td>Mac OSX Verison 2.0 or higher</td>
		</tr>
		<tr>
			<td>Gilson Pipetman</td>
			<td>Rainin instrument co. inc</td>
			<td></td>
			<td>P-20D, P-200D, P-1000D</td>
		</tr>
		<tr>
			<td>CK30/CK40 Culture Microscope</td>
			<td>Olympus</td>
			<td></td>
			<td>4 objective inverted light microscope with camera</td>
		</tr>
		<tr>
			<td>200 uL Pipet tips</td>
			<td>MidSci</td>
			<td>40200C</td>
			<td></td>
		</tr>
		<tr>
			<td>1000 uL Pipet tips</td>
			<td>MidSci</td>
			<td>AVR4</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL Seriological Pipets</td>
			<td>TPP</td>
			<td>TP94010</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well plates</td>
			<td>CoStar- Tissue Culture Cluster</td>
			<td align="right">3524</td>
			<td>24 wells, 16 mm well diameter, Radiation sterilized</td>
		</tr>
		<tr>
			<td>Trypan Blue Solution 0.4%</td>
			<td>Sigma</td>
			<td>T8154</td>
			<td>100 mL, cell culture tested non-haz</td>
		</tr>
		<tr>
			<td>Bright-line Hemacytometer replacement coverslip, non-haz</td>
			<td>Sigma</td>
			<td>Z375357</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Mammary Tumor(MMT) cells</td>
			<td>ATCC</td>
			<td>CCL-51</td>
			<td></td>
		</tr>
		<tr>
			<td>Eagle Minimum Essentail Medium (EMEM)</td>
			<td>ATCC</td>
			<td>30-2003</td>
			<td>500 mL</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Sigma</td>
			<td>F0926</td>
			<td>500 mL</td>
		</tr>
		<tr>
			<td>Meformin Hydrochloride</td>
			<td>Sigma</td>
			<td>PHR1084</td>
			<td>500 mg</td>
		</tr>
		<tr>
			<td>Tamoxifen</td>
			<td>Sigma</td>
			<td>T5648</td>
			<td>white or white-yellow powder</td>
		</tr>
		<tr>
			<td>Curmumin</td>
			<td>Sigma</td>
			<td>C1386</td>
			<td>yellow-orange powder</td>
		</tr>
		<tr>
			<td>Aspirin</td>
			<td>Sigma</td>
			<td>A2093</td>
			<td>meets USP testing specifications</td>
		</tr>
	</tbody>
</table>

using mouse mammary tumor cells to teach core biology concepts: a simple lab module

Undergraduate biology students are required to learn, understand and apply a variety of cellular and molecular biology concepts and techniques in preparation for biomedical, graduate and professional programs or careers in science. To address this, a simple laboratory module was devised to teach the concepts of cell division, cellular communication and cancer through the application of animal cell culture techniques. Here the mouse mammary tumor (MMT) cell line is used to model for breast cancer. Students learn to grow and characterize these animal cells in culture and test the effects of traditional and non-traditional chemotherapy agents on cell proliferation. Specifically, students determine the optimal cell concentration for plating and growing cells, learn how to prepare and dilute drug solutions, identify the best dosage and treatment time course of the antiproliferative agents, and ascertain the rate of cell death in response to various treatments. The module employs both a standard cell counting technique using a hemocytometer and a novel cell counting method using microscopy software. The experimental procedure lends to open-ended inquiry as students can modify critical steps of the protocol, including testing homeopathic agents and over-the-counter drugs. In short, this lab module requires students to use the scientific process to apply their knowledge of the cell cycle, cellular signaling pathways, cancer and modes of treatment, all while developing an array of laboratory skills including cell culture and analysis of experimental data not routinely taught in the undergraduate classroom.

גידול תאי MMT והשוואת שיטות ספירה. תאי גידול החלב עכבר גדלו בהצלחה ומאופיינת (איור 1) ושיטת ספירת תאים חדשנית שפותחה באמצעות MOTIC תוכנה, הקשורים מצלמה דיגיטלי תוכנה למיקרוסקופ. שיטת ספירת תאים חדשה זו בהשוואה לשיטת ספי?...

Watch this Scientific Journal Video about Using Mouse Mammary Tumor Cells to Teach Core Biology Concepts: A Simple Lab Module at JoVE.com

שימוש בתאי גידול חלבי בעכבר כדי ללמד מושגי ביולוגיה | ביולוגיה | וִידֵאוֹ

Using Mouse Mammary Tumor Cells to Teach Core Biology Concepts: A Simple Lab Module

A feasible laboratory module for biology undergraduates that explores advanced cellular and molecular concepts using animal cell culture is described. Students grow, characterize and manipulate a breast cancer cell model by exposure to chemotherapy agents. Cell viability is assayed through cell counting using both a standard and novel method.

שימוש בתאים סרטניים עכבר החלב ללמד מושגי ביולוגיה Core: מודול מעבדה פשוט

Undergraduate biology students are required to learn, understand and apply a variety of cellular and molecular biology concepts and techniques in ...

using-mouse-mammary-tumor-cells-to-teach-core-biology-concepts-simple

Research

Education

JoVE Journal

JoVE Core

Biology

Fundamentals

Scientific Inquiry

SCIENTISTS IN ACTION

12.8K Views.  Marymount Manhattan College. A feasible laboratory module for biology undergraduates that explores advanced cellular and molecular concepts using animal cell culture is described. Students grow, characterize and manipulate a breast cancer cell model by exposure to chemotherapy agents. Cell viability is assayed through cell counting using both a standard and novel method.

Video: Using Mouse Mammary Tumor Cells to Teach Core Biology Concepts: A Simple Lab Module

Visualizing Antigen Specific CD4+ T Cells using MHC Class II Tetramers

Major histocompatibility complex (MHC) class II tetramers allow the direct visualization of antigen specific CD4+ T cells by flow cytometry.  This method relies on the highly specific interaction between peptide loaded MHC and the corresponding T-cell receptor.  While the affinity of a single MHC/peptide molecule is low, cross-linking MHC/peptide complexes with streptavidin increases the avidity of the interaction, enabling their use as staining reagents.  Because of the relatively low frequencies of CD4+ T cells (~1 in 300,000 for a single specificity) this assay utilizes an in vitro amplification step to increase its threshold of detection.  Mononuclear cells are purified from peripheral blood by Ficoll underlay.  CD4+ cells are then separated by negative selection using biotinylated antibody cocktail and anti-biotin labeled magnetic beads.  Using adherent cells from the CD4- cell fraction as antigen presenting cells, CD4+ T cells are expanded in media by adding an antigenic peptide and IL-2.  The expanded cells are stained with the corresponding class II tetramer by incubating at 37 C for one hour and subsequently stained using surface antibodies such as anti-CD4, anti-CD3, and anti-CD25.  After labeling, the cells can be directly analyzed by flow cytometry.  The tetramer positive cells typically form a distinct population among the expanded CD4+ cells.  Tetramer positive cells are usually CD25+ and often CD4 high.  Because the level of background tetramer staining can vary, positive staining results should always be compared to the staining of the same cells with an irrelevant tetramer.  Multiple variations of this basic assay are possible.  Tetramer positive cells may be sorted for further phenotypic analysis, inclusion in ELISPOT or proliferation assays, or other secondary assays.  Several groups have also demonstrated co-staining using tetramers and either anti-cytokine or anti-FoxP3 antibodies.  

This procedure demonstrates the purification and in vitro expansion of antigen specific CD4+ T cells from whole peripheral blood and their visualization using MHC class II tetramers. Tetramers permit the direct visualization of T cells with a single antigen specificity and defined MHC class II restriction.

Antigen CD4 + הדמיה ספציפיות תאים T באמצעות MHC Class II Tetramers

Major histocompatibility complex (MHC) class II tetramers allow the direct visualization of antigen specific CD4+ T cells by flow cytometry.  This method ...

Mouse Mammary Epithelial Cells form Mammospheres During Lactogenic Differentiation

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of dome shaped cell structures referred to as mammospheres 1. The HC11 cell line has been employed as a model system for the study of regulation of mammary lactogenic differentiation both in vitro and in vivo 2. The HC11 cells differentiate and synthesize milk proteins in response to treatment with lactogenic hormones. Following the growth of HC11 mouse mammary epithelial cells to confluence, lactogenic differentiation was induced by the addition of a combination of lactogenic hormones including dexamethasone, insulin, and prolactin, referred to as DIP. The HC11 cells induced to differentiate were photographed at times up to 120 hours post induction of differentiation and the number of mammospheres that appeared in each culture was enumerated. The size of the individual mammospheres correlates with the degree of differentiation and this is depicted in the images of the differentiating cells.

HC11 lactogenic differentiation can be characterized by the formation of domed structures referred to as mammospheres. The structures can be enumerated by phase contrast microscopy to aid in quantifying lactogenic differentiation.

תאים אפיתל עכבר החלב טופס Mammospheres במהלך התמיינות Lactogenic

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of ...

Mammary Epithelial Transplant Procedure

This article describes and compares the fat pad clearance procedure developed by DeOme KB et al.1 and the sparing procedure developed by Brill B et al.2, followed by the mammary epithelial transplant procedure. The mammary transplant procedure is widely used by mammary biologists because it takes advantage of the fact that significant development of the mammary epithelium doesn't occur until after puberty. At 3 weeks of age, growth of the mammary epithelial tree is confined to the vicinity of the nipple and the fat pad is largely devoid of mammary epithelium, but by 7 weeks of age the epithelial ductal tree extends throughout the entire fat pad. Therefore, if this small portion of the fat pad containing epithelium, the region between the nipple and the lymph node, is removed at 3 weeks of age, the endogenous epithelium will never populate the mammary fat pad and the fat pad is described as "cleared". At this time, mammary epithelium from another source can be transplanted in the cleared fat pad where it has the potential to extend mammary ductal trees through out the fat pad. This procedure has been utilized in many experimental models including the examination of tumor phenotype in transgenic mammary epithelial tissue without the confounding effects of genotype on the entire animal3, in the identification of mammary stem cells by transplanting cells in limited dilution4,5, determining if hyperplastic nodules proceed to mammary tumors6, and to assess the effect of prior hormone exposure on the behavior of the mammary epithelium7,8.
Three week old host mice are anesthetized, cleaned and restrained on a surgical stage. A mid-sagittal incision is made through the skin, but not the peritoneum, extending from the pubis to the sternum. Oblique cuts are made through the skin from the mid-sagittal incision across the pelvis toward each leg. The skin is pulled away from the peritoneum to expose the 4th inguinal mammary gland. The fat pad is cleared by removing the fat pad tissue anterior to the lymph node. Epithelium fragments or epithelial cells are transplanted into the remaining cleared fat pad and the mouse is closed.

This article demonstrates the procedure developed by DeOme KB et al. (1959) and the sparing procedure developed by Brill B et al. (2008) for clearing the 4th inguinal mammary fat pad of a pubescent mouse in preparation for transplantation of mammary fragments, mammary epithelial cells, or mammary tumor cells.

החלב להשתלות אפיתל נוהל

This article describes and compares the fat pad clearance procedure developed by DeOme KB et al.1 and the sparing procedure developed by Brill B et al.2, ...

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 ...

Isolation of Immune Cells from Primary Tumors

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various chemokines and growth factors 1,2. However, once in the TME, these cells lose the ability to promote anti-tumor immunity and begin to support tumor growth and down-regulate anti-tumor immune responses 3-4. Studies on tumor-associated leukocytes have mainly focused on cells isolated from tumor-draining lymph nodes or spleen due to the inherent difficulties in obtaining sufficient cell numbers and purity from the primary tumor. While identifying the mechanisms of cell activation and trafficking through the lymphatic system of tumor bearing mice is important and may give insight to the kinetics of immune responses to cancer, in our experience, many leukocytes, including dendritic cells (DCs), in tumor-draining lymph nodes have a different phenotype than those that infiltrate tumors 5,6 . Furthermore, we have previously demonstrated that adoptively-transferred T cells isolated from the tumor-draining lymph nodes are not tolerized and are capable of responding to secondary stimulation in vitro unlike T cells isolated from the TME, which are tolerized and incapable of proliferation or cytokine production 7,8. Interestingly, we have shown that changing the tumor microenvironment, such as providing CD4+ T helper cells via adoptive transfer, promotes CD8+ T cells to maintain pro-inflammatory effector functions 5. The results from each of the previously mentioned studies demonstrate the importance of measuring cellular responses from TME-infiltrating immune cells as opposed to cells that remain in the periphery. To study the function of immune cells which infiltrate tumors using the Miltenyi Biotech isolation system9, we have modified and optimized this antibody-based isolation procedure to obtain highly enriched populations of antigen presenting cells and tumor antigen-specific cytotoxic T lymphocytes. The protocol includes a detailed dissection of murine prostate tissue from a spontaneous prostate tumor model (TRansgenic Adenocarcinoma of the Mouse Prostate -TRAMP) 10 and a subcutaneous melanoma (B16) tumor model followed by subsequent purification of various leukocyte populations.

In this report, we describe a protocol for isolating highly purified populations of leukocytes that infiltrate tumors. This protocol is adapted from the Miltenyi Biotech protocol to enhance yield and purity for isolating cells from complex tumor tissue.

בידוד של תאים חיסוניים של גידולים ראשוניים

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

דור של עכברים שמקורם בתאי גזע pluripotent מושרים

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Intraductal Injection for Localized Drug Delivery to the Mouse Mammary Gland

Herein we describe a protocol to deliver various reagents to the mouse mammary gland via intraductal injections. Localized drug delivery and knock-down of genes within the mammary epithelium has been difficult to achieve due to the lack of appropriate targeting molecules that are independent of developmental stages such as pregnancy and lactation. Herein, we describe a technique for localized delivery of reagents to the mammary gland at any stage in adulthood via intraductal injection into the nipples of mice. The injections can be performed on live mice, under anesthesia, and allow for a non-invasive and localized drug delivery to the mammary gland. Furthermore, the injections can be repeated over several months without damaging the nipple. Vital dyes such as Evans Blue are very helpful to learn the technique. Upon intraductal injection of the blue dye, the entire ductal tree becomes visible to the eye. Furthermore, fluorescently labeled reagents also allow for visualization and distribution within the mammary gland. This technique is adaptable for a variety of compounds including siRNA, chemotherapeutic agents, and small molecules.

A protocol for the non-invasive intraductal delivery of aqueous reagents to the mouse mammary gland is described. The method takes advantage of localized injection into the nipples of mammary glands targeting mammary ducts specifically. This technique is adaptable for a variety of compounds including siRNA, chemotherapeutic agents and small molecules.

הזרקת intraductal עבור משלוח סמים מקומיים לעכבר החלב בלוטת

Herein we describe a protocol to deliver various reagents to the mouse mammary gland via intraductal injections. Localized drug delivery and knock-down of ...

In vivo Reprogramming of Adult Somatic Cells to Pluripotency by Overexpression of Yamanaka Factors

Induced pluripotent stem (iPS) cells that result from the reprogramming of somatic cells to a pluripotent state by forced expression of defined factors are offering new opportunities for regenerative medicine. Such clinical applications of iPS cells have been limited so far, mainly due to the poor efficiency of the existing reprogramming methodologies and the risk of the generated iPS cells to form tumors upon implantation.
We hypothesized that the reprogramming of somatic cells towards pluripotency could be achieved in vivo by gene transfer of reprogramming factors. In order to efficiently reprogram cells in vivo, high levels of the Yamanaka (OKSM) transcription factors need to be expressed at the target tissue. This can be achieved by using different viral or nonviral gene vectors depending on the target tissue. In this particular study, hydrodynamic tail-vein (HTV) injection of plasmid DNA was used to deliver the OKSM factors to mouse hepatocytes. This provided proof-of-evidence of in vivo reprogramming of adult, somatic cells towards a pluripotent state with high efficiency and fast kinetics. Furthermore no tumor or teratoma formation was observed in situ.
It can be concluded that reprogramming somatic cells in vivo may offer a potential approach to induce enhanced pluripotency rapidly, efficiently, and safely compared to in vitro performed protocols and can be applied to different tissue types in the future.

In vivo Reprogramming of Adult Somatic Cells to Pluripotency by Overexpression of Yamanaka Factors

This study demonstrates the reprogramming of somatic cells towards pluripotency in vivo without the generation of teratomas. We used hydrodynamic tail vein injection of plasmid DNA encoding the Yamanka factors to induce the in vivo reprogramming of adult hepatocytes into cells of enhanced pluripotency.

בתכנות מחדש vivo של תאים סומטיים למבוגרים לpluripotency ידי ביטוי יתר של גורמי יאמאנאקה

Induced pluripotent stem (iPS) cells that result from the reprogramming of somatic cells to a pluripotent state by forced expression of defined factors ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

Nephrotoxin Microinjection דג הזברה למודל אי-ספיקת כליות חריפה

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

Measuring Lactase Enzymatic Activity in the Teaching Lab

Understanding how enzymes work, and relating this to real life examples, is critical to a wide range of undergraduate degrees in the biological and biomedical sciences. This easy to follow protocol was developed for first year undergraduate pharmacy students and provides an entry-level introduction to enzyme reactions and analytical procedures for enzyme analysis. The enzyme of choice is lactase, as this represents an example of a commercially available enzyme relevant to human disease/pharmaceutical practice. Lactase is extracted from dietary supplement tablets, and assessed using a colorimetric assay based upon hydrolysis of an artificial substrate for lactase (ortho-nitrophenol-beta-D-galactopyranoside, ONPG). Release of ortho-nitrophenol following the hydrolytic cleavage of ONPG by lactase is measured by a change in absorbance at 420 nm, and the effect of the temperature on the enzymatic reaction is evaluated by carrying out the reaction on ice, at room temperature and at 37 &#176;C. More advanced analysis can be implemented using this protocol by assessing the enzyme activity under different conditions and using different reagents.

The enzymatic activity of lactase is essential for the catabolic processing of the disaccharide lactose. Here, the activity of lactase found in dietary supplements is assayed using a colorimetric assay. This provides students with an experimental platform for understanding the activity of lactase and enzyme kinetics.

מדידת פעילות אנזימטי לקטאז במעבדת ההוראה

Understanding how enzymes work, and relating this to real life examples, is critical to a wide range of undergraduate degrees in the biological and ...