Serene Samyesudhas and Dr. Lynn Roy assisted in preparing samples for filming.

.1 תא תרבות ותיוג פלורסנט של שורות תאי סרטן השחלות <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכן תקשורת SKOV3: תקשורת מקוי בתוספת סרום 10% שור עוברי (FBS), L-גלוטמין 0.1 מ&quot;מ, 50 U / ml פניצילין, ו50 מיקרוגרם / מ&quot;ל ​​סטרפטומיצין. לשמור OVCA429 תאים בתקשורת המינימלי חיונית (MEM) בתוספת 10% FBS, פירובט נתרן 1 מ&quot;מ, L-גלוטמין 0.1 מ&quot;מ, 50 U / ml פניצילין, ו50 מיקרוגרם / מ&quot;ל ​​סטרפטומיצין. </li><li style=";text-align:right;direction:rtl"> הפץ שורות תאי SKOV3 וOVCA429 בחממה humidified על 37 מעלות צלזיוס עם 5% CO 2. לגדל תאים לconfluency 40-50%. </li><li style=";text-align:right;direction:rtl"> לייצר תאים לבטא חלבון אדום ניאון (RFP) במטרה לעקוב אחר כדאיות במבחנת in vivo. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> כדי ליצור תאי SKOV3 להביע RFP, להוסיף חלקיקי lentiviral להביע RFP וpolybrene / מ&quot;ל ​​10 מיקרוגרם לתקשורת SKOV3 בהעדר פניצילין וסטרפטומיצין ל72 שעה. </li><li style=";text-align:right;direction:rtl"> לאחר 72 שעה, לבחור עבור RFP תאים חיוביים על ידי תא הקרינה הופעל Sorting (FACS) כדלקמן: RFP Trypsinize ולא RFP לבטא בתאים, צנטריפוגות ב XG 125 למשך 5 דקות, תאים גלולים ב10 7 / מ&quot;ל תאי PBS המכיל BSA 0.1%. על סדרן תא, להשתמש בתאים שאינם RFP כדי לקבוע את הפרמטרים למיון. ואז לאסוף תאי המבטאים גבוה RFP באמצעות לייזר 561 ננומטר. </li><li style=";text-align:right;direction:rtl"> לחלופין, אם פלסמיד lentiviral מבטא קלטת עמידות לאנטיביוטיקה, לבחור עבור תאי RFP להביע ידי culturing באנטיביוטיקה המתאימה (כגון 10 מ&quot;ג / מ&quot;ל ​​blasticidin). הערה: הכמות של חלקיקי lentiviral שצריך להיות בשימוש יכול להשתנות על ידי אצווה ועל ידי סוג תא. כייל lentiviral נכון צריך להיות שנקבע לכל סוג תא. </li></ol></li></ol> .2 העשרה של תאי גזע סרטניים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> פנק את SKOV3, SKOV3-RFP, או OVCA429 שורות תאים עם 20 מיקרומטר של ציספלטין ל72 שעה. זהירות: ציספלטין הוא רעיל. השתמש בבגדים / כפפות מגן ולהימנע ממגע עם קרומי עור וריר. אל תשליך אני ציספלטיןשפכי n. </li><li style=";text-align:right;direction:rtl"> הכן תקשורת CSC: תקשורת DMEM / F12 סרום ללא תוספת 5 מיקרוגרם / מ&quot;ל ​​אינסולין, 10 ng / גורם גדילה באפידרמיס רקומביננטי אנושי מ&quot;ל (EGF), 10 ng / גורם גדילה פיברובלסטים בסיסי מ&quot;ל (bFGF), ng 12 / גורם מעכב לוקמיה מ&quot;ל , ואלבומין 0.4% בסרום שור (BSA). </li><li style=";text-align:right;direction:rtl"> תאי Trypsinize. תרבות ששרד תאים בתקשורת CSC. הערה: לאחר 2 ימים בspheroids שאינו חסיד התרבות יהוו. </li><li style=";text-align:right;direction:rtl"> שינוי בתקשורת כל 2 ימים על ידי איסוף מדיה המכילה תאים וצנטריפוגה ב125 XG למשך 5 דקות. אז resuspend גלולה בתקשורת CSC הטרי. בדקו תאים לכדאיות כל 2 ימים על ידי ספירת תאים וביצוע הרחקת trypan כחולה. </li><li style=";text-align:right;direction:rtl"> ואז לאסוף CSCS עבור ניסויים נוספים על ידי צנטריפוגה 129 XG למשך 5 דקות וresuspending גלולה בthiocynate פנול המכיל guanidinium (RNA), שנאגרו מלוח פוספט (PBS: 137 mM NaCl, 2.7 מ&quot;מ KCl, ו11.9 מ&quot;מ חיץ פוספט, pH 7.4) עם BSA 0.1% (עבור cytometry זרימה), או מדיה CSC להמשיךculturing התאים. </li><li style=";text-align:right;direction:rtl"> צג מורפולוגיה של תאי CSC באמצעות מיקרוסקופ אור ב20X ו40X הגדלה. </li></ol> .3 אפיון CSC באמצעות ניתוח ביטוי גנים לסמני תאי גזע <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לאסוף 3 הכנות של CSCS על ידי איסוף מדיה המכילה תאים, צנטריפוגה ב129 XG למשך 5 דקות, וresuspending גלולה בפתרון של thiocynate guanidinium פנול המכיל (ניתן להשתמש בשיטות אחרות להפקת RNA). </li><li style=";text-align:right;direction:rtl"> הכן RNA סך באמצעות TRIzol על פי הפרוטוקול של היצרן. לסנתז DNA המשלים (cDNA) .1-1 מיקרוגרם של RNA באמצעות ערכת cDNA ההפוך תמלול זמינה מסחרי. </li><li style=";text-align:right;direction:rtl"> לבצע תגובה הפוכה עיבד כמותית השרשרת של פולימראז (qRT-PCR). <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> מרכיבים את תערובת הורים עבור כל קבוצה של פריימרים המכילים תערובת qRT-PCR, פריימרים, ומים לסך 8 μl לדגימה (יכולים להיות מוגברים פריימרים עם fluorophores שונה באותה היטב). לדוגמא, ליצורתערובת אב אחד לOct4-FAM, Nanog-Hex, וGAPDH-Cy5. צור תערובות הורים נפרדות לNestin וProm1. הוסף 2 μl של תבנית cDNA לדגימה. הערה: במחקר זה, qRT-PCR בוצע באמצעות פריימרים מפורטים בטבלה 1. </li><li style=";text-align:right;direction:rtl"> לבצע את כל qRT-PCR בשני עותקים עבור כל דגימה באמצעות זמן אמת thermocycler PCR מסוגל לאתר fluorophores מרובה. כולל לא שולט תבנית. </li><li style=";text-align:right;direction:rtl"> לנרמל ביטוי גנים בתאי גזע לביטוי GAPDH עבור כל דגימה בשיטת ΔΔC T. </li></ol></li></ol><table border="0" cellpadding="0" cellspacing="0" width="746" style=";text-align:right;direction:rtl"><colgroup><col /><col /></colgroup><tbody><tr height="21"><td height="63" rowspan="3" style="height:62px;width:111px;"> אוקטובר -4-FAM </td><td style="width:636px;"> פריימר 1: 5&#39;-CCCAAGGAATAGTCTGTAGAAGTG-3 &quot; </td></tr><tr height="21"><td height="21" style="height:21px;"> פריימר 2: 5&#39;-TGCATGAGTCAGTGAACAGG-3 &quot;</td></tr><tr height="21"><td height="21" style="height:21px;"> בדיקה FAM: 5&#39;-CTTCCAAGC / ZEN / TGCCCACCTAACTTCT / 3IABkFQ -3 &quot; </td></tr><tr height="21"><td height="63" rowspan="3" style="height:62px;"> Nanog-HEX </td><td> פריימר 1: 5&#39;-CCTTCTGCGTCACACCATT-3 &quot; </td></tr><tr height="21"><td height="21" style="height:21px;"> פריימר 2: 5&#39;-AACTCTCCAACATCCTGAACC-3 &quot; </td></tr><tr height="21"><td height="21" style="height:21px;"> הבדיקה HEX: 5&#39;-CTGCCACCT / ZEN.CTTAGATTTCATTCTCTGGT / 3IABkFQ-3 &quot; </td></tr><tr height="21"><td height="63" rowspan="3" style="height:62px;"> GAPDH-Cy5 </td><td> פריימר 1: 5&#39;-TGTAGTTGAGGTCAATGAAGGG-3 &quot; </td></tr><tr height="21"><td height="21" style="height:21px;"> פריימר 2: 5&#39;-ACATCGCTCAGACACCATG-3 &quot; </td></tr><tr height="21"><td height="21" style="height:21px;"> הבדיקה Cy5: 5&#39;-AAGGTCGGAGTCAACGGATTTGGTC / 3IABRQSP-3 &quot; </td><html></tr><tr height="21"><td height="63" rowspan="3" style="height:62px;"> Nestin-FAM </td><td> פריימר 1: 5&#39;-AGGACCTGAGCGATCTGG-3 &quot; </td></tr><tr height="21"><td height="21" style="height:21px;"> פריימר 2: 5&#39;-CGTTGGAACAGAGGTTGGAG-3 &quot; </td></tr><tr height="21"><td height="21" style="height:21px;"> בדיקה FAM: 5&#39;-AACTTTTCA / ZEN / GTAGCCCGCAGCC / 3IABkFQ -3 &quot; </td></tr><tr height="21"><td height="63" rowspan="3" style="height:62px;"> CD133-HEX </td><td> פריימר 1: 5&#39;-ACTCTCTCCAACAATCCATTCC-3 &quot; </td></tr><tr height="21"><td height="21" style="height:21px;"> פריימר 2: 5&#39;-AAACAATTCACCAGCAACGAG-3 &quot; </td></tr><tr height="21"><td height="21" style="height:21px;"> הבדיקה HEX: 5&#39;-ACAATCACT / ZEN / GAGCACTCTATACCAAAGCG / 3IABkFQ-3 </td></tr></tbody></table> Primers הטבלה 1 המשמש לאפיון CSC ידי qRT-PCR. e_title &quot;&gt; 4. CSCS ניתוח לתא שטח סמני CD117 וCD133 <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> CSCS קציר על ידי איסוף מדיה המכילה תאים וצנטריפוגה ב125 XG למשך 5 דקות. הוסף 500 μl של פתרון ניתוק מסוג התאים לא פרוטאוליטים לשבור-up תאים. תאי צנטריפוגה ב125 XG למשך 5 דקות כדי להסיר פתרון ניתוק תא. לאחר מכן לשטוף את תאים פעמיים עם PBS הקר. </li><li style=";text-align:right;direction:rtl"> דגירה בתקשורת צמיחה נורמלית עבור שעה 1 לפני תאי תיוג. ספין למטה תאים 5 דקות ב129 x גרם. Resuspend תאים בPBS עם BSA 0.1% עם אנטי CD133-PE ואנטי CD117-FITC. </li><li style=";text-align:right;direction:rtl"> תקן תאי הלילה בparaformaldehyde 1%. זהירות: Paraformaldehyde הוא (קרצינוגן חשד) רעיל. הפוך במנדף ולהשתמש תוך שבוע. </li><li style=";text-align:right;direction:rtl"> לבצע cytometry זרימה לנתח CD117 (עם אנטי CD117 מצומדת לFITC) ביטוי על פני השטח של תאים בזרימה cytometer וCD133 (עם אנטי CD133 מצומדת לPE). לאסוף נתונים על תאים בFL1 (לFITC) וFL3 (לPE) ערוצים. צור שערים לתאים המבטאים את שניFITC וPE. צור עלילת נקודה עם 4 רביעים (FITC- וPE-, FITC + PE-, FITC- PE +, וFITC + וPE +) ולקבוע את מספר התאים בכל רבע. </li></ol> .5 ניתוח CSCS לתגובה טיפולית <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> צלחת 5,000 תאים לכל טוב (SKOV3, SKOV3-RFP, SKOV3 CSC וSKOV3-RFP CSCS) על צלחות 96 היטב הלילה. </li><li style=";text-align:right;direction:rtl"> פנקו עם ציספלטין (0, 1, 10, 100, ו1,000 מיקרומטר) או פקליטקסל (0, 0.01, 0.1, 1, 10, ו100 מיקרומטר) ל96 שעה. </li><li style=";text-align:right;direction:rtl"> בצע MTT (3 (4,5-Dimethylthiazol-2-י.ל.) ברומיד -2,5-diphenyltetrazolium) assay כדאיות תא. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> צלחת תאים 5,000 לכל טוב 100 μl על צלחת 96 היטב (תאי צלחת בשני עותקים וכוללים תקשורת לשלוט רק בארות). </li><li style=";text-align:right;direction:rtl"> לאחר 24 שעות, להוסיף 10 μl של 10 מ&quot;ג / מ&quot;ל ​​MTT. לדגור על 37 מעלות צלזיוס למשך 2-4 שעות (עד צורות משקע). </li><li style=";text-align:right;direction:rtl"> הוספה של MTT ממס 4 מ&quot;מ HCl 100 μl, 0.1% NP-40 בisopropanol. דגירה במשך 15 דקות בחושך. </li><li style=";text-align:right;direction:rtl"> קראו צלחות ב570 ננומטר על r צלחתeader. </li></ol></li></ol>

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The authors declare they have no competing financial interest.

CSCS כי הם עמידים לטיפול עשוי להיות אחראי להישנות לאחר הטיפול של גידול ראשוני. אפיון של CSCS עלול להוביל לטיפולים משופרים לסרטן השחלות. הפרמטרים הקריטיים בהקמת CSCS chemoresistant באמצעות הפרוטוקול לעיל תזמון משך טיפול עם כימותרפיה והריכוז של כימותרפיה. בעת שימוש בפרוטוקול בא...

Resistance to chemotherapy is a major impediment to the treatment and cure of cancer. Ovarian cancer is the 5th leading cause of cancer-related deaths among women in the United States (American Cancer Society Facts and Figures 2013). Patients initially respond well to chemotherapy, but most patients relapse1. After relapse patients are treated with a variety of additional chemotherapy agents with very little benefit2. General properties of CSCs include unlimited proliferative capabilities, retention of an undifferentiated state, resistance to drug treatment, efficient DNA repair, and ability to generate malignant tumor cells with different phenotypes3. CSCs frequently exhibit expression of stem cell genes such as Nanog, Oct4, Sox2, Nestin, CD133, and CD117. CSCs often express elevated levels of ABCG2, and ALDH genes that may contribute to drug efflux and metabolism3,4. 
The first definitive evidence for CSCs was demonstrated by isolating acute myeloid leukemia-initiating cells that were capable of self-renewal and tumor generation5. These leukemic stem cells expressed surface CD34 and generated leukemia in NOD/SCID (immunocompromised) mice5. Since then CSCs have been identified in many cancer types including leukemias/lymphomas, breast, bladder, colorectal, endometrial, sarcomas, hepatocellular carcinoma, melanomas, gliomas, ovarian, pancreatic, prostate, squamous cell carcinoma, and lung6. Therefore, being able to study this subtype of cancer cell is advantageous.
The goal of this study is to create a protocol for the selection and isolation of chemoresistant CSCs. Several methods have been reported for the isolation of CSCs from ovarian cancer cell lines. Non-adherent spheroids isolated from OVCAR-3, SKOV3, or HO8910 cultures demonstrate stem-like properties7,8. Isolation of CD133+ cells from OVCAR-3 cultures also yields CSCs. CSCs have also been selected in culture by treatment with chemotherapeutic agents. Treating tumor cell lines (OVCA433, Hey, and SKOV3 cells) with cisplatin and paclitaxel allows for the expansion and isolation of CSCs4,9. While culture of some cell types in CSC media leads to isolation of CSCs, SKOV3 cells did not survive culture in serum-free media or form sphere cells4. Therefore, treatment of cells with cisplatin and paclitaxel aided the expansion or isolation of this population4.
Using a modification of the procedure presented by Ma and colleagues4 we developed a method to isolate CSCs from the ovarian cancer cell lines. Our protocol is advantageous as it yields more viable cells while using less toxic chemotherapeutic agents. Cells are treated with cisplatin and subsequently grown in serum-free media supplemented with growth factors (stem cell media). We isolate the resulting non-adherent sphere cells and assay them for their expression of stem cell markers. This model enables the study of CSC properties and response to drug therapy.

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			<td height="43" style="height:43px;width:216px;">Name of Reagent/ Equipment</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">McCoy</td>
			<td style="width:71px;">Life Technologies</td>
			<td style="width:119px;">16600-108</td>
			<td style="width:167px;">Warm to 37C prior to use</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">DMEM / F12 serum free</td>
			<td style="width:71px;">Life Technologies&#160;</td>
			<td style="width:119px;">11320-033</td>
			<td style="width:167px;">Warm to 37C prior to use</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">Minimal Essential Media</td>
			<td style="width:71px;">Life Technologies&#160;</td>
			<td>42360032</td>
			<td style="width:167px;">Warm to 37C prior to use</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">Sodium Pyruvate</td>
			<td style="width:71px;">Life Technologies&#160;</td>
			<td><a href="http://www.lifetechnologies.com/order/catalog/product/11360070">11360070</a></td>
			<td style="width:167px;"></td>
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		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Polybrene</td>
			<td style="width:71px;">Millipore</td>
			<td>TR-1003-G</td>
			<td style="width:167px;"></td>
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		<tr height="61">
			<td height="61" style="height:61px;width:216px;">Blasticidin</td>
			<td style="width:71px;">Life Technologies&#160;</td>
			<td><a href="http://www.lifetechnologies.com/order/catalog/product/R21001">R21001</a></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">Fetal Bovine Serum&#160;</td>
			<td style="width:71px;">Atlas Biologicals</td>
			<td style="width:119px;">F-0500-A</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">Penicillin-streptomycin&#160;</td>
			<td style="width:71px;">Life Technologies</td>
			<td style="width:119px;">15070-063</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Cisplatin</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">T7402-5MG</td>
			<td style="width:167px;">Caution: Toxic Use precautions</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">pLenti-suCMV-Rsv</td>
			<td style="width:71px;">Gentarget</td>
			<td style="width:119px;">LVP023</td>
			<td style="width:167px;">BSL2 approval needed</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Insulin</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">I-1882</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="101">
			<td height="101" style="height:101px;width:216px;">Human Recombinant EGF&#160;</td>
			<td style="width:71px;">Cell Signaling Technology</td>
			<td style="width:119px;">8916LC</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">bFGF</td>
			<td style="width:71px;">BD biosciences</td>
			<td style="width:119px;">354060</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="41" rowspan="2" style="height:41px;width:216px;">LIF</td>
			<td rowspan="2" style="width:71px;">Santa Cruz</td>
			<td rowspan="2" style="width:119px;">sc-4988A</td>
			<td rowspan="2" style="width:167px;"></td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Bovine Serum Albumin</td>
			<td style="width:71px;">Roche</td>
			<td style="width:119px;">03 116 956 001</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">TRIzol</td>
			<td style="width:71px;">Life Technologies</td>
			<td style="width:119px;">15596-018</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">High Capacity cDNA Reverse Transcription Kit&#160;&#160;</td>
			<td style="width:71px;">Applied Biosystems</td>
			<td style="width:119px;">4368813</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">IQ Multiplex Powermix</td>
			<td style="width:71px;">BioRad</td>
			<td style="width:119px;">1725849</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Accumax</td>
			<td style="width:71px;">Millipore</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="101">
			<td height="101" style="height:101px;width:216px;">Primers</td>
			<td style="width:71px;">Integrated DNA Technology</td>
			<td style="width:119px;">individually designed and ordered (see protocol for sequnces)</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Anti-CD133 PE</td>
			<td style="width:71px;">Milenyl</td>
			<td>130-098-826</td>
			<td style="width:167px;">Primer/probe sets are light sensitive</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">CD117-Biotin</td>
			<td style="width:71px;">Miltenly</td>
			<td>130-098-570</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">AntiBiotin-FITC</td>
			<td style="width:71px;">Miltenly</td>
			<td>130-098-796</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">Paraformaldehyde</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td>P6148-1KG</td>
			<td style="width:167px;">Caution: Toxic always prepare in hood and make fresh.</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">Trypsin</td>
			<td style="width:71px;">Life Technologies</td>
			<td><a href="http://www.lifetechnologies.com/order/catalog/product/25300062">25300062</a></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">MTT (3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide)&#160;</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">25200-072</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">EVOS Fl Epifluorescence and Transmitted Light Microscope</td>
			<td>Advanced Microscopy Group</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Biorad CFX96 C1000 System</td>
			<td style="width:71px;">Biorad</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">Beckman Coulter FC500 Flow Cytometer&#160;</td>
			<td style="width:71px;">Beckman Coulter</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">Spectramax 340PC384&#160;</td>
			<td style="width:71px;">Molecular Devices</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

enrichment for chemoresistant ovarian cancer stem cells from human cell lines

תאי גזע סרטני (CSCS) מוגדרים כקבוצת משנה של רכיבה על אופניים איטיים ותאים שלא עברו התמיינות אשר מחלקים סימטרי ליצירת שגשוג מאוד, חודרני, ותאי גידול chemoresistant. לכן, CSCS הוא אוכלוסייה אטרקטיבית של תאים למקד טיפולי. CSCS הם חזו לתרום למספר סוגי סרטן כמו כולל אלו שבדם, המוח, ריאות, מערכת עיכול, ערמונית, והשחלה. בידוד והעשרת אוכלוסיית תאים סרטניים לCSCS תאפשר לחוקרים ללמוד את המאפיינים, גנטיקה, ותגובה הטיפולית של CSCS. אנחנו שנוצרנו פרוטוקול שמעשיר reproducibly לCSCS סרטן השחלות משורות תאי סרטן השחלות (SKOV3 וOVCA429). מטופלים שורות תאים עם 20 ציספלטין מיקרומטר במשך 3 ימים. תאים ששרדו מבודדים ותרבותיים בתקשורת של תאי גזע ללא סרום המכילה ציטוקינים וגורמי גדילה. אנו מדגימים העשרת CSCS המטוהר אלה על ידי ניתוח התאים המבודדים לknown תאי גזע סמני Oct4, Nanog, וProm1 (CD133) וביטוי בתא שטח של CD177 וCD133. תערוכת CSCS מוגברת chemoresistance. שיטה זו לבידודה של CSCS היא כלי שימושי לחקר תפקידה של CSCS בהישנות chemoresistance וגידול.

על מנת להוכיח כי אנו מבודדים CSCS משורות תאי סרטן השחלות אפיתל באמצעות טיפול ציספלטין, שרכשנו ראשון את תמונות של שורות התאים לפני טיפול ואחרי הבחירה. אנחנו השתמשנו במיקרוסקופ אור כדי ללכוד תמונות של חסיד (ללא טיפול) SKOV3 וOVCA429 תאים וSKOV3 וOVCA429 CSCS (איור 1). CSCS להופיע ?...

Watch this Scientific Journal Video about Enrichment for Chemoresistant Ovarian Cancer Stem Cells from Human Cell Lines at JoVE.com

Enrichment for Chemoresistant Ovarian Cancer Stem Cells from Human Cell Lines

תאי גזע סרטני (CSCS) הם מעורבים בהישנות גידול עקב chemoresistance. יש לנו אופטימיזציה פרוטוקול לבחירה והרחבת CSCS משורות תאי סרטן השחלות. על ידי טיפול בתאים עם ציספלטין כימותרפיות וculturing בתאי גזע קידום תקשורת אנו מעשירים לתרבויות CSC לא חסידות.

העשרה לתאי גזע Chemoresistant סרטן השחלות משורות תאים אנושיות

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, ...

enrichment-for-chemoresistant-ovarian-cancer-stem-cells-from-human

Research

JoVE Journal

Medicine

13.4K Views.  Indiana University School of Medicine.  תאי גזע סרטני (CSCS) הם מעורבים בהישנות גידול עקב chemoresistance. יש לנו אופטימיזציה פרוטוקול לבחירה והרחבת CSCS משורות תאי סרטן השחלות. על ידי טיפול בתאים עם ציספלטין כימותרפיות וculturing בתאי גזע קידום תקשורת אנו מעשירים לתרבויות CSC לא חסידות. 

Video: Enrichment for Chemoresistant Ovarian Cancer Stem Cells from Human Cell Lines

Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue

Identification of cardiomyocyte nuclei has been challenging in tissue sections as most strategies rely only on cytoplasmic marker proteins1. Rare events in cardiac myocytes such as proliferation and apoptosis require an accurate identification of cardiac myocyte nuclei to analyze cellular renewal in homeostasis and in pathological conditions2. Here, we provide a method to isolate cardiomyocyte nuclei from post mortem tissue by density sedimentation and immunolabeling with antibodies against pericentriolar material 1 (PCM-1) and subsequent flow cytometry sorting. This strategy allows a high throughput analysis and isolation with the advantage of working equally well on fresh tissue and frozen archival material. This makes it possible to study material already collected in biobanks. This technique is applicable and tested in a wide range of species and suitable for multiple downstream applications such as carbon-14 dating3, cell-cycle analysis4, visualization of thymidine analogues (e.g. BrdU and IdU)4, transcriptome and epigenetic analysis.

Cardiac nuclei are isolated via density sedimentation and immunolabeled with antibodies against pericentriolar material 1 (PCM-1) to identify and sort cardiomyocyte nuclei by flow cytometry.

הבידוד של גרעיני Cardiomyocyte מרקמות שלאחר המוות

Identification of cardiomyocyte nuclei has been challenging in tissue sections as most strategies rely only on cytoplasmic marker proteins1. Rare events ...

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

האדם תאים סרטניים נוירואנדוקריניים קווים כמו מודל תלת ממדי לחקר הטיפול האנושי גידול נוירואנדוקריניים

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

בידוד, אפיון והשוואתי התמיינות של תאי גזע אנושיים שיניים זולות נגזרה שיניים קבועות על ידי שימוש בשתי שיטות שונות

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

Time-lapse imaging can be used to compare behavior of cultured primary preneoplastic mammary epithelial cells derived from different genetically engineered mouse models of breast cancer. For example, time between cell divisions (cell lifetimes), apoptotic cell numbers, evolution of morphological changes, and mechanism of colony formation can be quantified and compared in cells carrying specific genetic lesions. Primary mammary epithelial cell cultures are generated from mammary glands without palpable tumor. Glands are carefully resected with clear separation from adjacent muscle, lymph nodes are removed, and single-cell suspensions of enriched mammary epithelial cells are generated by mincing mammary tissue followed by enzymatic dissociation and filtration. Single-cell suspensions are plated and placed directly under a microscope within an incubator chamber for live-cell imaging. Sixteen 650 &mu;m x 700 &mu;m fields in a 4x4 configuration from each well of a 6-well plate are imaged every 15 min for 5 days. Time-lapse images are examined directly to measure cellular behaviors that can include mechanism and frequency of cell colony formation within the first 24 hr of plating the cells (aggregation versus cell proliferation), incidence of apoptosis, and phasing of morphological changes. Single-cell tracking is used to generate cell fate maps for measurement of individual cell lifetimes and investigation of cell division patterns. Quantitative data are statistically analyzed to assess for significant differences in behavior correlated with specific genetic lesions.

Time-lapse imaging is used to assess behavior of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer risk to determine if there are correlations between specific behavioral parameters and distinct genetic lesions.

הדמית זמן לשגות של תאי אפיתל חלב Preneoplastic ראשוניים הנגזרת ממודלי עכבר מהונדס גנטי של סרטן השד

Time-lapse  imaging can be used to compare behavior of cultured primary preneoplastic  mammary epithelial cells derived from different genetically ...

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

תרבות מטופל נגזר סלולרי ובידוד של CD133<sup&gt; +</sup&gt; תאי גזע סרטני משוערים מלנומה

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

בידוד של תאי גזע סרטני מדגימות אדם סרטן הערמונית

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

Method for Obtaining Primary Ovarian Cancer Cells From Solid Specimens

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a valuable tool for understanding ovarian cancer, their use has many limitations. These include the lack of heterogeneity and the plethora of genetic alterations associated with extended in vitro passaging.&#160;Here we describe a method that allows for rapid establishment of primary ovarian cancer cells form solid clinical specimens collected at the time of surgery. The method consists of subjecting clinical specimens to enzymatic digestion for 30 min. The isolated cell suspension is allowed to grow and can be used for downstream application including drug screening. The advantage of primary ovarian cancer cell lines over established ovarian cancer cell lines is that they are representative of the original specific clinical specimens they are derived from and can be derived from different sites whether primary or metastatic ovarian cancer.

This study describes a detailed method for isolation and characterization of primary ovarian cancer cells from solid clinical specimens. Ovarian cancer clinical specimens are subjected to enzymatic digestion to obtain viable, fibroblast-free epithelial ovarian cancer (EOC) cells highly suitable for downstream applications.

שיטה לקבלת תאי סרטן השחלות ראשיים מתוך דגימות מוצקים

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a ...

Evaluation of Stem Cell Properties in Human Ovarian Carcinoma Cells Using Multi and Single Cell-based Spheres Assays

Years of research indicates that ovarian cancers harbor a heterogeneous mixture of cells including a subpopulation of so-called &#8220;cancer stem cells&#8221; (CSCs) responsible for tumor initiation, maintenance and relapse following conventional chemotherapies. Identification of ovarian CSCs is therefore an important goal. A commonly used method to assess CSC potential in vitro is the spheres assay in which cells are plated under non-adherent culture conditions in serum-free medium supplemented with growth factors and sphere formation is scored after a few days. Here, we review currently available protocols for human ovarian cancer spheres assays and perform a side-by-side analysis between commonly used multi cell-based assays and a more accurate system based on single cell plating. Our results indicate that both multi cell-based as well as single cell-based spheres assays can be used to investigate sphere formation in vitro. The more laborious and expensive single cell-based assays are more suitable for functional assessment of individual cells and lead to overall more accurate results while multi cell-based assays can be strongly influenced by the density of plated cells and require titration experiments upfront. Methylcellulose supplementation to multi cell-based assays can be effectively used to reduce mechanical artifacts.

In vitro spheres assays are commonly used to identify cancer stem cells. Here we compare single with multi cell-based spheres assays. The more laborious single cell-based assays or methylcellulose supplementation give more accurate results while multi cell-based assays performed in liquid medium can be highly influenced by cell density.

הערכה של מאפייני תא גזע בקרצינומה של תאי השחלות אדם באמצעות Multi ומבחני Spheres מבוסס תא בודדים

Years of research indicates that ovarian cancers harbor a heterogeneous mixture of cells including a subpopulation of so-called &#8220;cancer stem ...

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor initiation and/or relapse. Clarifying the mechanisms by which these tumor-initiating cells (TICs) support tumor formation and progression could lead to the development of clinically favorable therapies. Ovarian cancer is a heterogeneous and highly recurrent disease. Recent studies suggest TICs may play an important role in disease biology. We have identified culture conditions that enrich for TICs from ovarian cancer cell lines. Growing either adherent cells or non-adherent &#8216;floater&#8217; cells in a low attachment plate with serum free media in the presence of growth factors supports the propagation of ovarian cancer TICs with stem cell markers (CD133 and ALDH activity) and increased tumorigenicity without the need to physically separate the TICs from other cell types within the culture. Although the presence of floater cells is not common for all cell lines, this population of cells with innate low adherence may have high tumorigenic potential.Compared to adherent cells grown in the presence of serum, TICs readily form spheres, are significantly more tumorigenic in mice, and express putative stem cell markers. The conditions are easy to establish in a timely manner and can be used to study signaling pathways important for maintaining stem characteristics, and to identify drugs or combinations of drugs targeting TICs. The culture conditions described herein are applicable for a variety of ovarian cancer cells of epithelial origin and will be critical in providing new information about the role of TICs in tumor initiation, progression, and relapse.

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

Tumor-initiating cells (TICs) may represent a viable therapeutic target for the treatment of ovarian cancer, a highly recurrent and fatal disease. We present a protocol for culture conditions that enrich for this highly tumorigenic population of cells.

בהעשרת מבחנה של תאי ייזום-גידול סרטן השחלות

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor ...

Mammosphere Formation Assay from Human Breast Cancer Tissues and Cell Lines

Similar to healthy tissues, many blood and solid malignancies are now thought to be organised hierarchically, with a subset of stem-like cancer cells that self-renew while giving rise to more differentiated progeny. Understanding and targeting these cancer stem cells in breast cancer, which may possess enhanced chemo- and radio-resistance compared to the non-stem tumor bulk, has become an important research area. Markers including CD44, CD24, and ALDH activity can be assessed using fluorescence activated cell sorting (FACS) to prospectively isolate cells that display enhanced tumorigenicity when implanted into immunocompromised mice: the mammosphere assay has also become widely used for its ability to retrospectively identify sphere-forming cells that develop from single stem cell-like clones. Here we outline approaches for the appropriate culturing of mammospheres from cell lines or primary patient samples, their passaging, and calculations to estimate sphere forming efficiency (SFE). First we discuss key considerations and pitfalls in the appropriate planning and interpretation of mammosphere experiments.

Floating mammosphere assays can investigate the subset of stem-like breast cancer cells that survive in suspension conditions and show enhanced tumorigenesis when implanted into mice. This protocol provides a convenient in vitro measure of sphere-forming ability, a proxy for in vivo tumorigenesis, while facilitating analysis of the stem-associated transcriptional landscape.

Assay גיבוש Mammosphere משד אדם סרטן רקמות וקווים סלולריים

Similar to healthy tissues, many blood and solid malignancies are now thought to be organised hierarchically, with a subset of stem-like cancer cells that ...