עבודה זו נתמכה על ידי מענקי fromthe קרן בלגית למחקר מדעי (FNRS), Les Amis de l&#39;Institut Bordet, FNRS הפעולה Télévie, התכנית למלחמה בסרטן בבלגיה, Fonds לאמבו-Marteaux, Fonds JC Heuson וFonds Barsy.

הערה: כל הדגימות נרכשו באמצעות פרוטוקול שאושר על ידי ועדת האתיקה הרפואית של מכון ז&#39;ול בורדה בכתב הסכמה מהדעת שהתקבלה מכל מטופל. 1. הכנת homogenate רקמות <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לנתח רקמות resected (רקמה ממארת ונורמלית שעברה כריתה מחדר הניתוח) נמצא במעבדה הפתולוגית על ידי צוות מיומן לאיסוף מיידי. גידול, Nant (נלקח המרחק הרחוק ביותר מהגידול ככל האפשר) ושברי רקמה נורמלים מעובדים בתוך 1 באופן שגרתי - 3 שעות של כריתה כירורגית במעבדה BSL2 באמצעות נהלי בטיחות ביולוגיים סטנדרטיים לרקמות אנושיות. תרשים זרימה של הפרוטוקול באיור 1. </li><li style=";text-align:right;direction:rtl"> לשקול את כל שברי הרקמה (רגיל, Nant וגידול) ולמדוד את האורך, רוחב, וגובה (גובה x אורך x רוחב). זהו צעד חשוב לנורמליזציה הבאה של תת-אוכלוסיות של תאים, RNA שחולץ, וכו &#39; NOTE: מגוון רחב של גודל מדגם הוא בין 100 ל -10,000 מ&quot;מ 3 ללא שומן במידת האפשר. </li><li style=";text-align:right;direction:rtl"> חותם בר הגידול בשקופית זכוכית לצביעת H &amp; E כדי לוודא שהרקמה היא למעשה חלק של הגידול. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לעשות זאת על ידי לחיצה על שקופית מיקרוסקופ זכוכית על בר הגידול והפעלת לחץ עדין עם האצבעות שלך לכמה שניות. </li><li style=";text-align:right;direction:rtl"> תקן את השקופיות עם isopropanol למשך 2 דקות ואחריו צעד כביסה במים. Counterstain הרקמה למשך 30 שניות עם hematoxylin של מאיר. </li><li style=";text-align:right;direction:rtl"> לשטוף את השקופית בשש אמבטיות של מים. כתם בPhloxine B 2% למשך 15 שניות. </li><li style=";text-align:right;direction:rtl"> לשטוף באמבטיה של מים אחד ואחריו ארבע אמבטיות של isopropanol ולסיים עם אמבטיה אחת של מים. </li><li style=";text-align:right;direction:rtl"> דגירה בisopropanol דקות 1 וניקוז. ברור בשתי אמבטיות של קסילן. </li><li style=";text-align:right;direction:rtl"> הר עם הרכבה בינונית מבוסס קסילן. לבחון ל- תאי גידול (איור 2). הערה: בעיקר, תאים סרטניים לדבוק שקופית המוטבעות - סטרומה, הלימפה או מודעהipose תאים רק לעתים נדירות נותרו, ולהשאיר רווחים בין התאים הסרטניים (איור 2). </li></ol></li><li style=";text-align:right;direction:rtl"> מניחים את בר הרקמה בצלחת תרבות קטנה המכילה 1 מיליליטר של הגדרה כימית, המדיום הסלולרי hematopoietic סרום ללא (להלן כבינוני) בטמפרטורת חדר לקוביות אותו לחתיכות קטנות (~ 1 - 2 מ&quot;מ 2) באמצעות סטרילי אזמל. </li><li style=";text-align:right;direction:rtl"> העבר את הכל (שברי רקמה + בינוני) לצינור C Dissociator מכאני. </li><li style=";text-align:right;direction:rtl"> יש לשטוף את צלחת פטרי ואזמל עם 2 מיליליטר של מדיום בעזרת פיפטה פסטר ולהוסיף את זה לצינור C (נפח בינוני לניתוק = 3 מיליליטר). </li><li style=";text-align:right;direction:rtl"> השתמש בA.01 המכנית תכנית Dissociator לצינורות C (התכנית העדינה ביותר) לhomogenize ברי הרקמה לתוך השעיה תא בודדת. מניחים את צינור C במנגנון ולהפעיל את התכנית פעמיים ברציפות (מחזור אחד = 25 שניות). הערה: הליך הומוגניזציה זה כבר נקבע ואושר לרקמת שד אנושית, קיבה אחרתאו או רקמת סוגים ייתכן שיצטרכו להשתמש בתכנית אחרת וצריכים להיבדק ראשון. </li><li style=";text-align:right;direction:rtl"> הסר את צינור C מהמנגנון ולמזוג homogenate ישירות לתוך מסננת תא 40 מיקרומטר יושבת על צינור 50 מיליליטר. שימוש באותה פיפטה פסטר כמו בשלב 1.6, להעביר כל נוזל שנותר בצינור ג &#39;למסננת התא. </li><li style=";text-align:right;direction:rtl"> העבר את הנוזל המסונן לתוך צינור 15 מיליליטר באמצעות קצה micropipette 1 מיליליטר. באופן זמני לשמור את מסננת התא ושפופרת 50 מיליליטר שלה. </li><li style=";text-align:right;direction:rtl"> יש לשטוף את צינור C עם 3 מיליליטר נוסף של מדיום ולהעביר את זה, שוב באמצעות אותו פיפטה פסטר כמו בשלב 1.6, למסננת התא עדיין יושבת על צינור 50 מיליליטר. לסחוט סכום מקסימאלי של הנוזל שיורית לכודים ברקמות unhomogenized לתוך צינור 50 מיליליטר בעדינות על ידי הזזתו סביב המסננת עם טפטפת נקי פסטר או טיפ 1 מיליליטר שנזרק משם בהמשך, כדי למנוע זיהום eluate. </li><li style=";text-align:right;direction:rtl"> מניחים את מסננת התא במהופך על tהוא צינור מקורי C ולשטוף עם 3 מיליליטר של מדיום, כך שרקמת unhomogenized הטיפות בחזרה לתוך צינור C. </li><li style=";text-align:right;direction:rtl"> -Homogenize מחדש כמו בשלב 1.7 לשני מחזורים של תכנית A.01. </li><li style=";text-align:right;direction:rtl"> יוצקים homogenate השני זה דרך מסננת התא יושבת על צינור 50 מיליליטר, לשטוף את צינור C שוב עם 3 בינוני מיליליטר (כמו בשלב 1.10) ולהעביר עם פיפטה פסטר למסננת התא יושבת על צינור 50 מיליליטר שוב לוחץ מרבי כמות הנוזל מרקמת חיבור שיורית לכודה במסננת התא. </li><li style=";text-align:right;direction:rtl"> בשלב זה, בהיקף של ~ 2.5 מיליליטר הוא בצינור 15 מ&quot;ל ו~ 9 מיליליטר בשפופרת 50 מיליליטר. </li></ol> 2. הפרדה של רקמות ותאי Supernatant <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> צנטריפוגה homogenates ב -15 מיליליטר ו -50 מיליליטר צינורות במשך 15 דקות ב 600 XG בטמפרטורת חדר. </li><li style=";text-align:right;direction:rtl"> למזוג SN מצינור 15 מיליליטר לתוך צינור נקי ובאופן זמני לאחסן על 4 מעלות צלזיוס. supernatant זה = גידול ראשוני, Nant או ti הרגילssue SN (נפח סופי של 2.5 מיליליטר) יובהר בהמשך וaliquoted לפני האחסון ב -80 ° C עבור ניתוחים בעתיד (ראה להלן). </li><li style=";text-align:right;direction:rtl"> בטל supernatant מצינור 50 מיליליטר. </li><li style=";text-align:right;direction:rtl"> בעדינות resuspend שני כדורי התא בנפח סופי של 1 מיליליטר בינוני. בקצרה, ראשון לשבור בעדינות התא גלולה בשני הצינורות (על ידי הקשה על הצינור על משטח קשה). Resuspend תא גלולה הרופפת במ&quot;ל 50 עם 500 μl של מדיום ולהעביר השעיה תא זה לצינור 15 מיליליטר לresuspend גלולה השני. חזור על שלב זה פעם אחת עם 500 μl השני של מדיום להתאוששות המרבית של תאים. </li><li style=";text-align:right;direction:rtl"> העברת 10 μl של ההשעיה תא צינור קטן, לערבב עם 10 μl של trypan כחול (דילול של 1: 1) ולספור את מספר תאי קיימא באמצעות hemocytometer. הערה: בשלב זה כל חלק של ההשעיה התא יכול גם להיות מנותח על ידי הזרימה cytometry כדי להעריך גודל תא, גרעיניות ואם ארצה במספר מוגבל של סמנים תת-אוכלוסייה ליותר PreCהערכת ISE של חלוקת התא היחסית בhomogenate לפני ניתוח או ניסויים נרחבים. כל הניתוחים על ידי זרימת cytometry לשלב תיוג CD45 לנורמליזציה של תת-אוכלוסיות. </li><li style=";text-align:right;direction:rtl"> גלולה התאים על ידי צנטריפוגה XG ב 300 במשך 10 דקות בטמפרטורת חדר. התאים מן הגידול, Nant, או רקמה נורמלית מוכנים לטיהור או ניתוח נוסף עכשיו. צעדים נוספים אלה הם הטובים ביותר כאשר היא מבוצעת באותו היום כניתוח. הערה: לניתוח תזרים cytometric אך לא תא מיון תאי דם האדומים שיורית יש lysed לאחר תיוג נוגדנים על ידי הוספת 0.4 מיליליטר של חיץ תמוגה תא דם האדום לתא גלולה, מייד vortexing 1 שניות ודוגרים על מינימום של 10 דקות בחדר טמפרטורה (מוגן מפני אור) לפני הניתוח. </li></ol> 3. הבהרה של Supernatant רקמות <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> צנטריפוגה הצינורות 1.5 מיליליטר עם SN הרקמה ב 15,000 XG במשך 15 דקות ב 4 מעלות צלזיוס. </li><li style=";text-align:right;direction:rtl"> להסיר בזהירות tהוא supernatant בלי לגעת או להפריע גלולה. מעביר צינור נקי (או צינורות) תלוי במספר ובהיקף של aliquots הרצוי. </li><li style=";text-align:right;direction:rtl"> אחסן את supernatant ב -80 ° C לשימוש עתידי. </li></ol> 4. דם חולה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לאסוף דגימת דם מכל מטופל כביקורת על ידי venipuncture לתוך צינורות heparinized היום לפני הניתוח. צנטריפוגה הדם ב 400 XG במשך 10 דקות ב 20 מעלות צלזיוס עם הבלם מלהשיג את הפלזמה ומעיל באפי. </li><li style=";text-align:right;direction:rtl"> הסר את הפלזמה ולהבהיר את זה על ידי צנטריפוגה ב10,000 XG במשך 15 דקות בטמפרטורה של 20 מעלות צלזיוס. Aliquot ופלזמה חנות ב -80 ° C לשימוש עתידי. לדלל את המעיל באפי במדיום </li><li style=";text-align:right;direction:rtl"> הפרד את תאי mononuclear באמצעות צנטריפוגה שיפוע ficoll-hypaque סטנדרטית לפני ניתוח מיידי, בידוד תת-אוכלוסייה, DNA / RNA / הפקת חלבון או הקפאה. </li></ol> 5. cytometry הזרימה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> תאי תווית על פי ייצורההוראות של r ב 4 ° C, מוגן מפני אור. תאי דם אדום בLyse השעיות תא מברי רקמה לאחר תיוג נוגדנים על ידי הוספת 0.4 מיליליטר של חיץ תמוגה תא לתא גלולה. </li><li style=";text-align:right;direction:rtl"> מערבולת מייד ל1 שניות ודגירת מינימום של 10 דקות בטמפרטורת חדר, מוגן מפני אור. להעביר את התאים שכותרתו בcytometer זרימה לרכישת נתונים ללא שטיפה. </li></ol>

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The authors declare that no conflict of interest exists.

מחקר זה מתאר שיטה מותאמת להכנה המהירה של homogenates רקמת שד הנורמלית וממאירים ללא עיכול אנזימטי למיון לאחר תא, חילוץ, הקפאה ו / או ניתוח פנוטיפי של CD45 + תת-אוכלוסיות. המטרה של הגישה הניסויית הזה היא לייצר תמונות של TIL המשקף באופן הדוק את מדינתם in vivo ולהשוות אותם לר...

The tumor microenvironment is composed of various cell types with numerous studies showing they each play distinct and important roles in tumorigenesis1,2. These include, but are not limited to, infiltrating immune cells, stromal cells, endothelial cells and tumor cells3. Ex vivo studies of tumor infiltrating lymphocytes (TIL; CD45+ cells or leukocytes, which are predominantly lymphocytes in breast tumors) from fresh human tissue samples is made difficult by their low frequency, the small sample sizes often available for research and the potential for loss of viability during extraction. Because immune cells infiltrating tumors are usually present as passengers rather than permanent residents in general they are easier to release from the tissue matrix.
Dissociating tumor tissue while maintaining cellular integrity is technically challenging and has traditionally been performed using a combination of mechanical and enzymatic steps to prepare single cell suspensions4-8. This approach involves lengthy incubation periods and is associated with a significant reduction in cell viability as well as the loss of cell surface receptors by enzymatic cleavage. High quality flow cytometric studies characterizing TIL in the tumor microenvironment as well as clean purifications of CD45+ subpopulations by flow cytometry or antibody-coated beads are more difficult to achieve from enzyme-digested tumor tissue. In addition, the supernatant (SN) from the resulting tumor homogenate is not amenable to further analysis including quantification of secreted proteins (cytokines, chemokines, immunoglobulins or tumor antigens) or experimental treatment of normal cells, because of the potential for protein degradation in the enzymatic digests.
In our search for a method to prepare single cell homogenates from breast tissues [including tumor, non-adjacent non-tumor (NANT) and normal (from mammary reductions) breast tissues] without enzymatic digestion, we tested a variety of mechanical homogenization techniques. Homogenates prepared using a mechanical dissociator had increased cell viability (2-fold) and total cell recovery (2-fold) while preserving surface receptor expression. Enzymatic digestion of the remaining insoluble material did not recover additional CD45+ cells suggesting they were all released in the initial homogenate. Thus, this rapid and simple approach allows both qualitative and quantitative assessment of the CD45+ subpopulations present in various normal and malignant human tissues. An added advantage of this approach is that the SN from the initial homogenate (primary tissue SN) can be collected and stored for further analysis or experimentation.

<table border="0" cellpadding="0" cellspacing="0" width="980">
	<tbody>
		<tr height="26">
			<td height="26" style="height:27px;width:272px;">Equipment</td>
			<td style="width:163px;">Company</td>
			<td style="width:137px;">Catalog Number</td>
			<td style="width:409px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">GentleMacs Dissociator&#160;</td>
			<td style="width:163px;">Miltenyi Biotec</td>
			<td style="width:137px;">130-093-235</td>
			<td>BD Medimachine is somewhat equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Centrifuge 5810 R</td>
			<td style="width:163px;">Eppendorf</td>
			<td style="width:137px;">N/A</td>
			<td>or other standard table top centrifuge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Centrifuge 5417 R</td>
			<td style="width:163px;">Eppendorf</td>
			<td style="width:137px;">N/A</td>
			<td>or other standard microcentrifuge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Esco Class II A2 Biosafety Cabinet</td>
			<td>ESCO global</td>
			<td style="width:137px;">N/A</td>
			<td>or other standard BSL2 hood</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Inverted Microscope</td>
			<td style="width:163px;">Nikon eclipse TS100</td>
			<td style="width:137px;">N/A</td>
			<td>or other microscope compatible for a hemacytometer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">B&#252;rker Chamber</td>
			<td style="width:163px;">Marienfield&#160;</td>
			<td style="width:137px;">640210</td>
			<td>or other standard hemacytometer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Navios Flow Cytometer</td>
			<td style="width:163px;">Beckman Coulter</td>
			<td style="width:137px;">N/A</td>
			<td>or other flow cytometer (8-10 color recommended)</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:272px;"></td>
			<td style="width:163px;"></td>
			<td style="width:137px;"></td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:272px;">Materials</td>
			<td style="width:163px;">Company</td>
			<td style="width:137px;">Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">GentleMacs C-Tube</td>
			<td style="width:163px;">Miltenyi Biotec</td>
			<td style="width:137px;">130-096-344</td>
			<td>BD Medimachine uses Filcon</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Cell Culture Dish</td>
			<td style="width:163px;">Sarstedt</td>
			<td style="width:137px;">72,710</td>
			<td>or other non-pyrogenic plasticware&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Disposable Scalpel</td>
			<td style="width:163px;">Swann-Morton</td>
			<td style="width:137px;">0510</td>
			<td>or standard single use sterile scalpel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">BD Cell Strainer 40&#181;m</td>
			<td style="width:163px;">Becton Dickinson</td>
			<td style="width:137px;">734-0002</td>
			<td>or other non-pyrogenic plasticware&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">BD Falcon Tube 50mL</td>
			<td style="width:163px;">Becton Dickinson</td>
			<td style="width:137px;">352070</td>
			<td>or other non-pyrogenic plasticware&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">BD Falcon Tube 15mL</td>
			<td style="width:163px;">Becton Dickinson</td>
			<td style="width:137px;">352097</td>
			<td>or other non-pyrogenic plasticware&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">BD FACS Tube 5mL</td>
			<td style="width:163px;">Becton Dickinson</td>
			<td style="width:137px;">352008</td>
			<td>or other non-pyrogenic plasticware&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Sterile Pasteur Pipette 5 mL&#160;</td>
			<td style="width:163px;">VWR</td>
			<td style="width:137px;">612-1685</td>
			<td>or other non-pyrogenic plasticware&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Microfuge Tube 1.5 mL</td>
			<td style="width:163px;">Eppendorf</td>
			<td style="width:137px;">7805-00</td>
			<td>or other non-pyrogenic plasticware&#160;</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:272px;"></td>
			<td style="width:163px;"></td>
			<td style="width:137px;"></td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:272px;">Reagents</td>
			<td style="width:163px;">Company</td>
			<td style="width:137px;">Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">X-Vivo 20</td>
			<td style="width:163px;">Lonza</td>
			<td style="width:137px;">BE04-448Q</td>
			<td>serum-free medium recommended</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Phosphate buffered saline</td>
			<td style="width:163px;">Lonza</td>
			<td style="width:137px;">BE17-516F</td>
			<td>standard physiological PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Trypan blue&#160;</td>
			<td style="width:163px;">VWR</td>
			<td style="width:137px;">17942E</td>
			<td>or other vital stain</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">VersaLyse</td>
			<td style="width:163px;">Beckman Coulter</td>
			<td style="width:137px;">A09777</td>
			<td>for flow cytometry experiments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Fixable viability Dye eFluor 780&#160;</td>
			<td style="width:163px;">eBioscience</td>
			<td style="width:137px;">65-0865-14</td>
			<td>for flow cytometry experiments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">anti-CD3 FITC</td>
			<td style="width:163px;">BD Biosciences</td>
			<td style="width:137px;">345763</td>
			<td>for flow cytometry experiments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">anti-CD3 Vio Blue</td>
			<td style="width:163px;">Miltenyi Biotec</td>
			<td style="width:137px;">130-094-363</td>
			<td>for flow cytometry experiments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">anti-CD4 PE</td>
			<td style="width:163px;">BD Biosciences</td>
			<td style="width:137px;">345769</td>
			<td>for flow cytometry experiments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">anti-CD4 APC</td>
			<td style="width:163px;">Miltenyi Biotec</td>
			<td style="width:137px;">130-091-232</td>
			<td>for flow cytometry experiments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">anti-CD8 ECD</td>
			<td style="width:163px;">Beckman Coulter</td>
			<td style="width:137px;">737659</td>
			<td>for flow cytometry experiments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">anti-CD8 PerCP</td>
			<td style="width:163px;">BD Biosciences</td>
			<td style="width:137px;">345774</td>
			<td>for flow cytometry experiments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">anti-CD19 APC-Vio770</td>
			<td style="width:163px;">Miltenyi Biotec</td>
			<td style="width:137px;">130-096-643</td>
			<td>for flow cytometry experiments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">anti-CD45 VioGreen</td>
			<td style="width:163px;">Miltenyi Biotec</td>
			<td style="width:137px;">130-096-906</td>
			<td>for flow cytometry experiments</td>
		</tr>
	</tbody>
</table>

a simple and rapid protocol to non-enzymatically dissociate fresh human tissues for the analysis of infiltrating lymphocytes

היכולת של תאים ממאירים ללהתחמק ממערכת החיסון, המתאפיינת בבריחת גידול משני תגובות חיסון מולדים ובעלי כושר הסתגלות, מקובל כיום כסימן היכר חשוב של הסרטן. המחקר שלנו על סרטן השד מתמקד בתפקיד הפעיל שלימפוציטים גידול הסתננות לשחק בהתקדמות גידול ותוצאת מטופל. לקראת מטרה זו, פיתחנו מתודולוגיה לבידוד המהיר של תאי הלימפה שלמים מרקמות נורמליות ולא נורמליות במאמץ כדי להעריך אותם בסמוך למדינה האם שלהם. Homogenates הוכנה באמצעות תכנית Dissociator מכאנית שתי התאוששות כדאיות ותא גדלה תוך שמירת רקמות לעומת ביטוי קולט משטח לאנזים-מעוכלות. יתר על כן, עיכול אנזימטי של החומר מסיס שנותר לא התאושש CD45 תאים + נוספים מצביעים על כך שמדידות כמותיות ואיכותיות בhomogenate העיקרי העשויים באמת משקפות תת-אוכלוסיות שחדרו בfragm הרקמהאף אוזן גרון. תאי הלימפה בhomogenates אלה יכולים להיות בקלות מאופיינת באמצעות חיסוני (פנוטיפ, הפצה, וכו &#39;) או מולקולרי (DNA, RNA ו / או חלבון) מתקרב. גם תאי CD45 + יכולים לשמש לטיהור תת-אוכלוסייה, התרחבות במבחנה או הקפאה. יתרון נוסף של גישה זו הוא שsupernatant הרקמה העיקרי מhomogenates יכול לשמש כדי לאפיין ולהשוות ציטוקינים, כמוקינים, נוגדנים ואנטיגנים הנמצאים ברקמות נורמליות וממאירים. פונקציות זה פרוטוקול טוב מאוד לרקמות שד אנושיות וצריכה להיות ישימות למגוון רחב של רקמות נורמליות ולא נורמליות.

עיכול אנזימתי של שברי רקמות עם שני פתרונות זמינים מסחרי ניתוק רקמה או תערובות מעבדה שונות של collagenase, DNase ו / או מעכבי hyaluronidase, לדבוק במגוון רחב של קולטנים על פני השטח של תאים. המחקרים שלנו, התמקדו בתחילה בתאי CD4 + T חדירת גידולים בשד, הוצגו במהירות עם בעיה טכנית עיקרי...

Watch this Scientific Journal Video about A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes at JoVE.com

A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes

This protocol describes the rapid non-enzymatic dissociation of fresh human tissue fragments for qualitative and quantitative assessment of CD45+ cells (lymphocytes/leukocytes) present in various normal and malignant human tissues. Additionally, the supernatant obtained from the primary tissue homogenate can be collected and stored for further analysis or experimentation.

פרוטוקול פשוט ומהיר לללא אנזימי רקמות אנושיות טריות לנתק לניתוח להחדרה לימפוציטים

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted ...

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Immunology and Infection

19.9K Views.  Université Libre de Bruxelles. This protocol describes the rapid non-enzymatic dissociation of fresh human tissue fragments for qualitative and quantitative assessment of CD45+ cells (lymphocytes/leukocytes) present in various normal and malignant human tissues. Additionally, the supernatant obtained from the primary tissue homogenate can be collected and stored for further analysis or experimentation.

Video: A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

שילוב של הדגימה דבק קלטת מבוססי פלואורסצנטי באתרו הכלאה עבור איתור מהיר של סלמונלה על תוצרת טרייה

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

Endemic countries are increasingly adopting molecular tools for efficient typing, identification and surveillance against malaria parasites and vector mosquitoes, as an integral part of their control programs1,2,3,4,5. For sustainable establishment of these accurate approaches in operations research to strengthen malaria control and elimination efforts, simple and affordable methods, with parsimonious reagent and equipment requirements are essential6,7,8. Here we present a simple Chelex-based technique for extracting malaria parasite and vector DNA from field collected mosquito specimens.
We morphologically identified 72 Anopheles gambiae sl. from 156 mosquitoes captured by pyrethrum spray catches in sleeping rooms of households within a 2,000 km2 vicinity of the Malaria Institute at Macha. After dissection to separate the head and thorax from the abdomen for all 72 Anopheles gambiae sl. mosquitoes, the two sections were individually placed in 1.5 ml microcentrifuge tubes and submerged in 20 &mu;l of deionized water. Using a sterile pipette tip, each mosquito section was separately homogenized to a uniform suspension in the deionized water. Of the ensuing homogenate from each mosquito section, 10 &mu;l was retained while the other 10 &mu;l was transferred to a separate autoclaved 1.5 ml tube. The separate aliquots were subjected to DNA extraction by either the simplified Chelex or the standard salting out extraction protocol9,10. The salting out protocol is so-called and widely used because it employs high salt concentrations in lieu of hazardous organic solvents (such as phenol and chloroform) for the protein precipitation step during DNA extraction9.
Extracts were used as templates for PCR amplification using primers targeting arthropod mitochondrial nicotinamide adenine dinucleotide dehydrogenase (NADH) subunit 4 gene (ND4) to check DNA quality11, a PCR for identification of Anopheles gambiae sibling species10 and a nested PCR for typing of Plasmodium falciparum infection12. Comparison using DNA quality (ND4) PCR showed 93% sensitivity and 82% specificity for the Chelex approach relative to the established salting out protocol. Corresponding values of sensitivity and specificity were 100% and 78%, respectively, using sibling species identification PCR and 92% and 80%, respectively for P. falciparum detection PCR. There were no significant differences in proportion of samples giving amplicon signal with the Chelex or the regular salting out protocol across all three PCR applications. The Chelex approach required three simple reagents and 37 min to complete, while the salting out protocol entailed 10 different reagents and 2 hr and 47 min' processing time, including an overnight step. Our results show that the Chelex method is comparable to the existing salting out extraction and can be substituted as a simple and sustainable approach in resource-limited settings where a constant reagent supply chain is often difficult to maintain.

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

A rapid and affordable way to extract quality malaria parasite and vector DNA from mosquito specimens is described. Capitalizing on chelating properties of Chelex resin, the simple method enables genotyping of malaria parasites in mosquito mid-gut and salivary gland phases, as well as molecular identification of the Anopheles sibling species by PCR.

פשוט פרוטוקול Chelex למיצוי DNA מ<em&gt; אנופלס</em&gt; Spp.

Endemic countries are increasingly adopting molecular tools for  efficient typing, identification and surveillance against malaria parasites and  vector ...

A Simple Protocol for Platelet-mediated Clumping of Plasmodium falciparum-infected Erythrocytes in a Resource Poor Setting

P. falciparum causes the majority of severe malarial infections. The pathophysiological mechanisms underlying cerebral malaria (CM) are not fully understood and several hypotheses have been put forward, including mechanical obstruction of microvessels by P. falciparum-parasitized red blood cells (pRBC). Indeed, during the intra-erythrocytic stage of its life cycle, P. falciparum has the unique ability to modify the surface of the infected erythrocyte by exporting surface antigens with varying adhesive properties onto the RBC membrane. This allows the sequestration of pRBC in multiple tissues and organs by adhesion to endothelial cells lining the microvasculature of post-capillary venules 1. By doing so, the mature forms of the parasite avoid splenic clearance of the deformed infected erythrocytes 2 and restrict their environment to a more favorable low oxygen pressure 3. As a consequence of this sequestration, it is only immature asexual parasites and gametocytes that can be detected in peripheral blood.
Cytoadherence and sequestration of mature pRBC to the numerous host receptors expressed on microvascular beds occurs in severe and uncomplicated disease. However, several lines of evidence suggest that only specific adhesive phenotypes are likely to be associated with severe pathological outcomes of malaria. One example of such specific host-parasite interactions has been demonstrated in vitro, where the ability of intercellular adhesion molecule-1 to support binding of pRBC with particular adhesive properties has been linked to development of cerebral malaria 4,5. The placenta has also been recognized as a site of preferential pRBC accumulation in malaria-infected pregnant women, with chondrotin sulphate A expressed on syncytiotrophoblasts that line the placental intervillous space as the main receptor 6. Rosetting of pRBC to uninfected erythrocytes via the complement receptor 1 (CD35)7,8 has also been associated with severe disease 9.
One of the most recently described P. falciparum cytoadherence phenotypes is the ability of the pRBC to form platelet-mediated clumps in vitro. The formation of such pRBC clumps requires CD36, a glycoprotein expressed on the surface of platelets. Another human receptor, gC1qR/HABP1/p32, expressed on diverse cell types including endothelial cells and platelets, has also been shown to facilitate pRBC adhesion on platelets to form clumps 10. Whether clumping occurs in vivo remains unclear, but it may account for the significant accumulation of platelets described in brain microvasculature of Malawian children who died from CM 11. In addition, the ability of clinical isolate cultures to clump in vitro was directly linked to the severity of disease in Malawian 12 and Mozambican patients 13, (although not in Malian 14).
With several aspects of the pRBC clumping phenotype poorly characterized, current studies on this subject have not followed a standardized procedure. This is an important issue because of the known high variability inherent in the assay 15. Here, we present a method for in vitro platelet-mediated clumping of P. falciparum with hopes that it will provide a platform for a consistent method for other groups and raise awareness of the limitations in investigating this phenotype in future studies. Being based in Malawi, we provide a protocol specifically designed for a limited resource setting, with the advantage that freshly collected clinical isolates can be examined for phenotype without need for cryopreservation.

A Simple Protocol for Platelet-mediated Clumping of Plasmodium falciparum-infected Erythrocytes in a Resource Poor Setting

This method investigates the platelet-mediated clumping phenotype of Plasmodium falciparum-infected erythrocytes (pRBC) in clinical isolates. This is performed by isolating and co-incubating platelet-rich plasma and a suspension of pRBC.

פרוטוקול פשוט לclumping טסיות דם בתיווך של<em&gt; Plasmodium falciparum</emאריתרוציטים&gt; נגועים בסביבה מסכנה משאבים

P. falciparum causes the majority of severe malarial infections. The pathophysiological mechanisms underlying cerebral malaria (CM) are not fully ...

Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency and type of chromosome aberrations in different cell types allows defects in DNA repair pathways to be elucidated. Understanding mammalian DNA repair biology has been greatly helped by the production of mice with knockouts in specific genes. The goal of this protocol is to quantify genomic instability in mouse B lymphocytes. Labeling of the telomeres using PNA-FISH probes (peptide nucleic acid - fluorescent in situ hybridization) facilitates the rapid analysis of genomic instability in metaphase chromosome spreads. B cells have specific advantages relative to fibroblasts, because they have normal ploidy and a higher mitotic index. Short-term culture of B cells therefore enables precise measurement of genomic instability in a primary cell population which is likely to have fewer secondary genetic mutations than what is typically found in transformed fibroblasts or patient cell lines.

DNA repair pathways are essential for maintenance of genomic integrity and preventing mutation and cancer. The goal of this protocol is to quantify genomic instability by direct observation of chromosome aberrations in metaphase spreads from mouse B cells using fluorescent in situ hybridization (FISH) for telomeric DNA repeats.

ניתוח מהיר של סטיות כרומוזום בעכבר לימפוציטים מסוג B על ידי הרשות הפלסטינית-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency ...

Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to the palatine tonsils situated at the lateral walls of the oral part of the pharynx. Surgically removed palatine tonsils provide a convenient accessible source of B and T lymphocytes to study the interplay between foreign pathogens and the host immune system. This video protocol describes the dissection and processing of surgically removed human palatine tonsils, followed by the isolation of the individual B and T cell populations from the same tissue sample. We present a method, which efficiently separates tonsillar B and T lymphocytes using an antibody-dependent affinity protocol. Further, we use the method to demonstrate that human adenovirus infects specifically the tonsillar T cell fraction. The established protocol is generally applicable to efficiently and rapidly isolate tonsillar B and T cell populations to study the role of different types of pathogens in tonsillar immune responses.

Palatine tonsils are a rich source of B and T lymphocytes. Here we provide an easy, efficient and rapid protocol to isolate B and T lymphocytes from human palatine tonsils. The method described has been specifically adapted for studies of the viral etiology of tonsil inflammation known as tonsillitis.

פרוטוקול בידוד יעיל עבור B ו- T לימפוציטים משקדי הפלטין אדם

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to ...

A Semi-automated Approach to Preparing Antibody Cocktails for Immunophenotypic Analysis of Human Peripheral Blood

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease and treatment. It has the potential to predict therapeutic efficacy and identify novel therapeutic targets. Whole blood staining utilizes unmanipulated blood, which minimizes artifacts that can occur during sample preparation. However, whole blood staining must also be done on freshly collected blood to ensure the integrity of the sample. Additionally, it is best to prepare antibody cocktails on the same day to avoid potential instability of tandem-dyes and prevent reagent interaction between brilliant violet dyes. Therefore, whole blood staining requires careful standardization to control for intra and inter-experimental variability.
Here, we report deployment of an automated liquid handler equipped with a two-dimensional (2D) barcode reader into a standard process of making antibody cocktails for flow cytometry. Antibodies were transferred into 2D barcoded tubes arranged in a 96 well format and their contents compiled in a database. The liquid handler could then locate the source antibody vials by referencing antibody names within the database. Our method eliminated tedious coordination for positioning of source antibody tubes. It provided versatility allowing the user to easily change any number of details in the antibody dispensing process such as specific antibody to use, volume, and destination by modifying the database without rewriting the scripting in the software method for each assay.
A proof of concept experiment achieved outstanding inter and intra- assay precision, demonstrated by replicate preparation of an 11-color, 17-antibody flow cytometry assay. These methodologies increased overall throughput for flow cytometry assays and facilitated daily preparation of the complex antibody cocktails required for the detailed phenotypic characterization of freshly collected anticoagulated peripheral blood.

Whole blood Immunophenotyping is indispensable for monitoring the human immune system. However, the number of reagents required and the instability of specific fluorochromes in premixed cocktails necessitates daily reagent preparation. Here we show that semi-automated preparation of staining antibody cocktail helps establish reliable immunophenotyping by minimizing variability in reagent dispensing.

גישה אוטומטית-למחצה הכנת קוקטיילי נוגדן עבור ניתוח Immunophenotypic של אדם דם היקפי

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease ...

A RAPID Method for Blood Processing to Increase the Yield of Plasma Peptide Levels in Human Blood

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the correct determination of a peptide&#39;s concentration, it should be stable during blood processing. However, this is not the case for several peptides which are quickly degraded by endogenous peptidases. Recently, we developed a blood processing method employing Reduced temperatures, Acidification, Protease inhibition, Isotopic exogenous controls and Dilution (RAPID) for the use in rats. Here, we have established this technique for the use in humans and investigated recovery, molecular form and circulating concentration of food intake regulatory hormones. The RAPID method significantly improved the recovery for 125I-labeled somatostatin-28 (+39%), glucagon-like peptide-1 (+35%), acyl ghrelin and glucagon (+32%), insulin and kisspeptin (+29%), nesfatin-1 (+28%), leptin (+21%) and peptide YY3-36 (+19%) compared to standard processing (EDTA blood on ice, p&#160;&#60;0.001). High performance liquid chromatography showed the elution of endogenous acyl ghrelin at the expected position after RAPID processing, while after standard processing 62% of acyl ghrelin were degraded resulting in an earlier peak likely representing desacyl ghrelin. After RAPID processing the acyl/desacyl ghrelin ratio in blood of normal weight subjects was 1:3 compared to 1:23 following standard processing (p&#160;= 0.03). Also endogenous kisspeptin levels were higher after RAPID compared to standard processing (+99%, p&#160;= 0.02). The RAPID blood processing method can be used in humans, yields higher peptide levels and allows for assessment of the correct molecular form.

The RAPID blood processing method can be used in humans and yields higher peptide levels as well as allows for assessment of the correct molecular form. Therefore, this method will be a valuable tool in peptide research.

שיטה מהירה עבור עיבוד דם כדי להגדיל את התשואה של רמות הפפטיד פלזמה בדם אנושי

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.

גישה פשוטה ויעילה לבנות וקטורי וירוס Vaccinia Mutant

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

An Optimized Protocol to Analyze Glycolysis and Mitochondrial Respiration in Lymphocytes

Lymphocytes respond to a variety of stimuli by activating intracellular signaling pathways, which in turn leads to rapid cellular proliferation, migration and differentiation, and cytokine production. All of these events are tightly linked to the energy status of the cell, and therefore studying the energy-producing pathways may give clues about the overall functionality of these cells. The extracellular flux analyzer is a commonly used device for evaluating the performance of glycolysis and mitochondrial respiration in many cell types. This system has been used to study immune cells in a few published reports, yet a comprehensive protocol optimized particularly for lymphocytes is lacking. Lymphocytes are fragile cells that survive poorly in ex vivo conditions. Oftentimes lymphocyte subsets are rare, and working with low cell numbers is inevitable. Thus, an experimental strategy that addresses these difficulties is required. Here, we provide a protocol that allows for rapid isolation of viable lymphocytes from lymphoid tissues, and for the analysis of their metabolic states in the extracellular flux analyzer. Furthermore, we provide results of experiments in which the metabolic activities of several lymphocyte subtypes at different cell densities were compared. These observations suggest that our protocol can be used to achieve consistent, well-standardized results even at low cell concentrations, and thus it may have broad applications in future studies focusing on the characterization of metabolic events in immune cells.

The study of metabolism is becoming increasingly relevant to immunological research. Here, we present an optimized method for measuring glycolysis and mitochondrial respiration in mouse splenocytes, and T and B lymphocytes.

פרוטוקול אופטימיזציה כדי לנתח הגליקוליזה מיטוכונדריאלי הנשימה לימפוציטים

Lymphocytes respond to a variety of stimuli by activating intracellular signaling pathways, which in turn leads to rapid cellular proliferation, migration ...

Rapid Quantification of Mitogen-induced Blastogenesis in T Lymphocytes for Identifying Immunomodulatory Drugs

Lymphocyte proliferation in response to antigenic or mitogenic stimulation is a readily quantifiable phenomenon useful for testing immunomodulatory (i.e., immunosuppressive or immunostimulatory) chemical compounds and biologics. One of the earliest steps during mitogenesis is cell enlargement or blastogenic transformation, whereupon the cell volume increases before division. It is usually detectable in the first several hours of T-lymphocyte stimulation. Here, we describe a rapid method to quantify blastogenesis in T lymphocytes isolated from mouse spleens and human peripheral blood mononuclear cells (PBMCs) using an automated cell counter. Various commonly used proliferation assays for the most part are laborious and only reflect the overall population effect rather than individual cellular effects within a population. In contrast, the presented automated cell counter assay provides rapid, direct, and precise measurements of cell diameters that can be used for assessing the effectiveness of various mitogens and immunomodulatory drugs in vitro.

T-lymphocyte mitogenesis is accompanied by blastogenic transformation, whereupon the cell volume enlarges before cell division. Here, we describe a method to quantify blastogenesis in T lymphocytes using an automated cell counter with the capability of measuring cell diameters.

כימות מהיר של Blastogenesis mitogen-Induced T לימפוציטים לזיהוי סמים המערכת החיסונית

Lymphocyte proliferation in response to antigenic or mitogenic stimulation is a readily quantifiable phenomenon useful for testing immunomodulatory (i.e., ...