Umbilical cords were collected with the assistance of the Birmingham Women's Health Care NHS Trust. HMM was supported by an Arthritis Research UK Career Development Fellowship (19899) and Systems Science for Health, University of Birmingham (5212).

<ol>
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M., Mannori, G., Cecconi, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial-leukocyte+adhesion+molecules+in+human+disease.">Endothelial-leukocyte adhesion molecules in human disease.</a> Annual review of medicine. 45, 361-378 (1994).</li><li>Stanness, K. A., Beatty, P. G., Ochs, H. D., Harlan, 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=An+endothelial+cell+surface+factor(s)+induced+in+vitro.">An endothelial cell surface factor(s) induced in vitro.</a> 136 (12), 4548-4553 (1986).</li><li>Burton, V. J., Butler, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delay+of+migrating+leukocytes+by+the+basement+membrane+deposited+by+endothelial+cells+in+long-term+culture.">Delay of migrating leukocytes by the basement membrane deposited by endothelial cells in long-term culture.</a> Experimental cell research. 317 (3), 276-292 (2011).</li><li>Luu, N. T., Rainger, G. E., Buckley, C. D., Nash, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD31+Regulates+Direction+and+Rate+of+Neutrophil+Migration+over+and+under+Endothelial+Cells.">CD31 Regulates Direction and Rate of Neutrophil Migration over and under Endothelial Cells.</a> Journal of Vascular Research. 40 (5), 467-479 (2003).</li><li>McGettrick, H. M., Buckley, C. D., Ed Rainger, G., Nash, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+stromal+cells+on+lymphocyte+adhesion+and+migration+on+endothelial+cells.">Influence of stromal cells on lymphocyte adhesion and migration on endothelial cells.</a> Methods in molecular biology. 616, 49-68 (2010).</li><li>Butler, L. M., McGettrick, H. M., Nash, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Static+and+dynamic+assays+of+cell+adhesion+relevant+to+the+vasculature.">Static and dynamic assays of cell adhesion relevant to the vasculature.</a> Methods in molecular biology. 467, 211-228 (2009).</li><li>Jeffery, H. C., Buckley, C. D., Moss, P., Rainger, G. E., Nash, G. B., McGettrick, H. 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The authors declare that they have no conflicts of interest.

כאן אנו מתארים שני מודלים במבחנה &quot;כלי דם&quot; ללימוד הגיוס של מחזורי נויטרופילים ידי האנדותל מודלק. יתרון עיקרי של מודלים אלה הוא היכולת לנתח כל צעד במפל ההידבקות לויקוציטים במטרה, כפי שהיה קורה בvivo. יש לנו שנצפה קודם לכן עלייה תלויה-מינון בהידבקות נויטרופ?...

דלקת היא תגובה הגנתית לזיהום חיידקים או פגיעה ברקמות שדורשת רגולציה הדוקה של כניסה לויקוציטים וליציאה מהרקמה הדלקתית לאפשר 1,2 רזולוציה. צלב-שיחה בין תאי האנדותל (EC) שכלי דם קו, כדוריות דם לבנות במחזור ותאי סטרומה רקמות תושב חיונית לתיאום תהליך זה 3. עם זאת, גיוס בלתי מבוקר של כדוריות דם לבנות והפינוי יעיל שלהם לחזק את התפתחותן של מחלות דלקתיות כרוניות 4. ההבנה הנוכחית שלנו של גיוס לויקוציטים בבריאות ובמחלה אינה שלמה ויש צורך במודלים חזקים יותר כדי לנתח את התהליך הזה. המנגנונים התומכים בגיוס של לויקוציטים מהדם דרך כלי דם בEC venules פוסט-נימים תוארו גם 1,2,5. כדוריות דם לבנות במחזור נלכדים על ידי קולטנים מיוחדים (למשל, VCAM-1, E-selectin, P-selectin) שהם מוסדרים על האנדותל מודלק. אינטראקציות חולפות אלה מאפשרים לויקוציטים לאינטראקציה עם כמוקינים משטח מחויב ומתווכים הנגזרים שומנים (או אנדותל או סטרומה במקור) המפעילים integrins לידי ביטוי על ידי כדוריות דם לבנות 6 11. זה בתורו מייצב הגירת הידבקות וכוננים על פני האנדותל ולתוך הרקמה 12 15. בתוך רקמה, גויס לויקוציטים נתונים לסוכנים הנגזר סטרומה המשפיעים על תנועתיות, תפקוד והישרדות 16,17. ראיות הולכות ומצטברים מצביעות בבירור על אותות המתקבלים בכל שלב של לויקוציטים תנאי תהליך הגיוס למשנו. עם זאת, ההבנה של גיוס לויקוציטים שלנו נשארת שלמה ומעט מאוד ידוע על תנועת ויקוציטים עיצוב רכיבים בתוך רקמה. בברמינגהם פיתחנו כמה מודלים &quot;כלי דם&quot; מבחנה ללמוד הגיוס של לויקוציטים מלזרום 9,18,19. עכשיו אנו מבינים כי רגולטורים מיידיים כמעשה EC כלי דם של גיוס לויקוציטים בתגובה לשינויים במייקרו-הסביבה המקומית שלהם. באופן ספציפי, תאי סטרומה רקמה-תושב יכולים להסדיר באופן פעיל את התגובה הדלקתית, בחלקו על ידי שיחה עם EC כלי הדם השכן להשפיע תפקידם בגיוס 3. הראינו בעבר כי תאי סטרומה שונים לווסת את היכולת של EC לתמוך הידבקות והגירה של לויקוציטים באופן רקמות ספציפיות, וכי ההשפעות אלה הופכים שינו במחלות כרוניות 13,16,20,21. כך, תאי סטרומה להקים רקמה &#39;כתובת קודים &quot;המגדירים את ההקשר של כל תגובה דלקתית 22. לאחרונה, יש לנו הפגנו שMSC מח עצם נגזר (BMMSC) מטה-להסדיר בעצמת התגובה של EC לציטוקינים, שהביא לירידה בגיוס של שני נויטרופילים וימפוציטים 23. מנגנוני גובגיוס erning הובהר במבחנה השתמש בעיקר מבחני שילוב סוג תא בודד (למשל, EC) או חלבון בבידוד. עם זאת, מחקרים אלה לא לוקחים בחשבון את ההשפעות של סביבת הרקמות המקומית (כלומר, הנוכחות של תאי סטרומה) ​​על גיוס של לויקוציטים וההגירה הבאה שלהם לתוך הרקמה. כאן אנו מתארים שתי שיטות המבוססת על זרימה שבו תאי סטרומה, במיוחד mesenchymal תאי גזע (MSC), הם שיתוף תרבותי עם EC 23. מודלים כאלה מאפשרים לנו לבחון את ההשפעה של תא סטרומה על תגובות אנדותל, ביכולת שלהם בפרט לתמוך גיוס לויקוציטים מזרימה.

<table border="0" cellpadding="0" cellspacing="0" >
	<tbody>
		<tr >
			<td >Collagenase Type Ia</td>
			<td >Sigma</td>
			<td>C2674</td>
			<td>Dilute in 10ml PBS to get a final concentration of 10mg/ml. Store at -20&#176;C in 1ml aliquots.</td>
		</tr>
		<tr >
			<td >Dulbecco&#39;s PBS</td>
			<td >Sigma</td>
			<td >D8662</td>
			<td>With calcium and magnesium chloride. Keep sterile and store at room temperature.</td>
		</tr>
		<tr >
			<td >1X Medium M199</td>
			<td >Gibco</td>
			<td >31150-022</td>
			<td>Warm in 37 &#176;C water bath before use.</td>
		</tr>
		<tr >
			<td >Gentamicin sulphate</td>
			<td >Sigma</td>
			<td >G1397</td>
			<td>Store at 4&#176;C. Add to M199 500ml bottle.</td>
		</tr>
		<tr >
			<td >Human epidermal growth factor</td>
			<td >Sigma</td>
			<td >E9644</td>
			<td>Store at -20&#176;C in 10&#181;l aliquots.</td>
		</tr>
		<tr >
			<td >Fetal calf serum (FCS)</td>
			<td >Sigma</td>
			<td >F9665</td>
			<td >FCS must be batch tested to ensure the growth and viability of isolated EC. Heat inactivate at 56&#176;C. Store in 10ml aliquots at -20&#176;C.</td>
		</tr>
		<tr >
			<td >Amphotericin B</td>
			<td >Gibco</td>
			<td >15290-026</td>
			<td >Potent and becomes toxic within a week so fresh complete HUVEC medium must be made up every week. Store at -20&#176;C in 1ml aliquots.</td>
		</tr>
		<tr >
			<td >Hydrocortisone</td>
			<td >Sigma</td>
			<td >H0135</td>
			<td>Stock is in ethanol. Store at -20&#176;C in 10&#181;l aliquots.</td>
		</tr>
		<tr >
			<td >Collagenase Type II</td>
			<td >Sigma</td>
			<td >C6885</td>
			<td>Dilute stock in PBS to a final concentration of 100mg/ml. Store at -20&#176;C in 100&#181;l aliquots.</td>
		</tr>
		<tr >
			<td >Hyaluronidase</td>
			<td >Sigma</td>
			<td >H3631</td>
			<td>Dilute stock in PBS to a final concentration of 20,000U/ml. Store at -20&#176;C in 100&#181;l aliquots.</td>
		</tr>
		<tr >
			<td >100&#181;m cell strainer for 50ml centifuge tube</td>
			<td >Scientific Lab Supplies (SLS)</td>
			<td >352360</td>
			<td >Other commercially available cell strainers (e.g. Greiner bio-one) can also be used.</td>
		</tr>
		<tr >
			<td >DMEM low glucose</td>
			<td >Biosera</td>
			<td >LM-D1102/500</td>
			<td>Warm in 37 &#176;C water bath before use.</td>
		</tr>
		<tr >
			<td >Penicillin/Streptomycin mix</td>
			<td >Sigma</td>
			<td >P4333</td>
			<td>Store at -20&#176;C in 1ml aliquots.</td>
		</tr>
		<tr >
			<td >25cm2 tissue culture flask</td>
			<td >SLS</td>
			<td >353109</td>
			<td></td>
		</tr>
		<tr >
			<td >75cm2 tissue culture flask</td>
			<td >SLS</td>
			<td >353136</td>
			<td></td>
		</tr>
		<tr >
			<td >Bone marrow mesenchymal stem cells vial</td>
			<td >Lonza</td>
			<td >PT-2501</td>
			<td >Store in liquid nitrogen upon arrival. Cells are at passage 2 upon arrival but are designated passage 0. Exapand to passage 3 and store in liquid nitrogen for later use.</td>
		</tr>
		<tr >
			<td >Mesenchymal stem cell growth medium (MSCGM)</td>
			<td >Lonza</td>
			<td >PT-3001</td>
			<td>Warm in 37 &#176;C water bath before use. For Cell Tracker Green staining use medium without FCS.</td>
		</tr>
		<tr >
			<td >EDTA (0.02%) solution</td>
			<td >Sigma</td>
			<td >E8008</td>
			<td>Store at 4&#176;C. Warm in 37&#176;C water bath before use.</td>
		</tr>
		<tr >
			<td >Trypsin solution</td>
			<td >Sigma</td>
			<td >T4424</td>
			<td>Store at -20&#176;C in 2ml aliquots. Thaw at room temperature and use immediately.</td>
		</tr>
		<tr >
			<td >Cryovials</td>
			<td >Greiner bio-one</td>
			<td>2019-02</td>
			<td>Keep on ice before adding before adding cell suspension.</td>
		</tr>
		<tr >
			<td >Mr. Frosty Freezing Container</td>
			<td >Nalgene</td>
			<td >5100-0001</td>
			<td >Store at room temperature. When adding cryovials with cells store at -80&#176;C for 24h before transfrring cells to liquid nitrogen.</td>
		</tr>
		<tr >
			<td >Ibidi u-Slide VI (0.4), T/C treated, sterile</td>
			<td >Ibidi</td>
			<td >IB-80606</td>
			<td >Alternative models include glass capillaries, Cellix Biochips (www.cellixltd.com), BioFlux Plates (www.fluxionbio.com/bioflux/) and GlycoTech parallel plate flow chambers (http://www.glycotech.com/apparatus/parallel.html).</td>
		</tr>
		<tr >
			<td >Cell tracker green dye</td>
			<td >Life technologies</td>
			<td >C2925</td>
			<td>Store in 5&#181;l aliquots at -20&#176;C. Dilute in 5ml prewarmed (at 37&#176;C) MSCGM.</td>
		</tr>
		<tr >
			<td >Cell counting chambers</td>
			<td >Nexcelom</td>
			<td >SD-100</td>
			<td>Alternatively a haemocytometer can be used.</td>
		</tr>
		<tr >
			<td >Cellometer auto T4 cell counter</td>
			<td >Nexcelom</td>
			<td >Auto T4-203-0238</td>
			<td></td>
		</tr>
		<tr >
			<td >Tumor necrosis factor &#945; (TNF&#945;)</td>
			<td >R&#38;D Systems</td>
			<td >210-TA-100</td>
			<td>Dilute stock in PBS to a final concentration of 100,000U/ml. Store at -80&#176;C in 10&#181;l aliquots.</td>
		</tr>
		<tr >
			<td >6-well, 0.4&#181;m PET Transwell filters</td>
			<td >SLS</td>
			<td >353090</td>
			<td></td>
		</tr>
		<tr >
			<td >K2-EDTA in 10ml tubes</td>
			<td >Sarstedt</td>
			<td ></td>
			<td>Store at room temperature.</td>
		</tr>
		<tr >
			<td >Histopaque 1119</td>
			<td >Sigma</td>
			<td >11191</td>
			<td>Store at 4&#176;C. Warm to room temperature before use.</td>
		</tr>
		<tr >
			<td >Histopaque 1077</td>
			<td >Sigma</td>
			<td >10771</td>
			<td>Store at 4&#176;C. Warm to room temperature before use.</td>
		</tr>
		<tr >
			<td >10ml round bottomed tube</td>
			<td >Appleton Woods</td>
			<td >SC211 142 AS</td>
			<td></td>
		</tr>
		<tr >
			<td >7.5% BSA Fraction V solution</td>
			<td >Life technologies</td>
			<td >15260-037</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr >
			<td >20ml Plastipak syringes</td>
			<td >BD falcon</td>
			<td >300613</td>
			<td></td>
		</tr>
		<tr >
			<td >5ml Plastipak syringes</td>
			<td >BD falcon</td>
			<td >302187</td>
			<td></td>
		</tr>
		<tr >
			<td >2ml Plastipak syringes</td>
			<td >BD falcon</td>
			<td >300185</td>
			<td></td>
		</tr>
		<tr >
			<td >3M hypo-allergenic surgical tape 9m x 2.5cm</td>
			<td >Micropore</td>
			<td >1530-1</td>
			<td>Use to secure the syringe tap onto the wall of the perspex chamber.</td>
		</tr>
		<tr >
			<td >Silicon rubber tubing, internal diameter/external diameter (ID/OD) of 1/3mm (thin tubing)</td>
			<td >Fisher Scientific</td>
			<td >FB68854</td>
			<td >Cut silicon tubing to the appropriate size. All tubing leading directly to the electronic microvalve must be thin.</td>
		</tr>
		<tr >
			<td >Silicon rubber tubing ID/OD of 2/4mm (thick tubing)</td>
			<td >Fisher Scientific</td>
			<td >FB68855</td>
			<td></td>
		</tr>
		<tr >
			<td >Portex Blue Line Manometer tubing</td>
			<td>Smiths</td>
			<td >200/495/200</td>
			<td>Tubing leading to the syringe pump.</td>
		</tr>
		<tr >
			<td >3-way stopcock</td>
			<td >BOC Ohmeda AB</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Glass 50ml syringe for pump</td>
			<td >Popper Micromate</td>
			<td >550962</td>
			<td>Must be primed prior to use by removing any air bubbles.</td>
		</tr>
		<tr >
			<td >Glass coverslip</td>
			<td >Raymond A Lamb</td>
			<td ></td>
			<td>26x76mm coverslips made to order. Lot number 2440980.</td>
		</tr>
		<tr >
			<td >Parafilm gasket</td>
			<td >American National Can Company</td>
			<td ></td>
			<td >Cut a 26x76mm piece of parafilm using an aluminium template and cut a 20x4mm slot into it using a scalpel 10a. Gasket thickness is approximately 133&#181;m.</td>
		</tr>
		<tr >
			<td >Two perspex parallel plates</td>
			<td >Wolfson Applied Technology Laboratory</td>
			<td ></td>
			<td >Specially designed chamber consisting of parallel plates held together by 8 screws. The lower plate has a viewing slot cut out in the middle and a shallow recess milled to allow space for the coverslip, filter and gasket. The upper perspex plate has an inlet and outlet hole positioned over the flow channel.</td>
		</tr>
		<tr >
			<td >Electronic 3-way microvalve with min. dead space</td>
			<td >Lee Products Ltd.</td>
			<td >LFYA1226032H</td>
			<td>Electronically connected to a 12 volt DC power supply.</td>
		</tr>
		<tr >
			<td >Syringe pump for infusion/withdrawal (PHD2000)</td>
			<td >Harvard Apparatus</td>
			<td >70-2001</td>
			<td>Set the diameter to 29mm and refill (flow) rate.</td>
		</tr>
		<tr >
			<td >L-shaped connector</td>
			<td >Labhut</td>
			<td >LE876</td>
			<td>To attach to the inlet and outlet ports onto the Ibidi microslide channel.</td>
		</tr>
		<tr >
			<td >Video camera</td>
			<td >Qimaging</td>
			<td >01-QIC-F-M-12-C</td>
			<td>Connected to a computer which enables digitall videos to be recorded.</td>
		</tr>
		<tr >
			<td >Image-Pro Plus 7.0</td>
			<td >Media Cybernetics</td>
			<td >41N70000-61592</td>
			<td >For data analysis. Manually tag cells displaying the different behaviors. Track cells for analysis of rolling and migration velocities.</td>
		</tr>
		<tr >
			<td ></td>
			<td ></td>
			<td ></td>
			<td>Refer to product datasheets for details on hazards of using the reagents described here.</td>
		</tr>
	</tbody>
</table>

analyzing the effects of stromal cells on the recruitment of leukocytes from flow

תאי סטרומה להסדיר את הגיוס במחזור leukocytes במהלך דלקת דרך הצטלבות עם תאי האנדותל שכנים. כאן אנו מתארים שני מודלים במבחנה &quot;כלי דם&quot; ללימוד הגיוס של מחזורי נויטרופילים מזרימה על ידי תאי האנדותל מודלקים. יתרון עיקרי של מודלים אלה הוא היכולת לנתח כל צעד במפל ההידבקות לויקוציטים במטרה, כפי שהיה קורה בvivo. אנו גם מתארים כיצד ניתן להתאים את שני המודלים כדי ללמוד את התפקיד של תאי סטרומה, בתאי גזע mesenchymal זה מקרה (MSC), בויסות גיוס לויקוציטים. תאי האנדותל עיקריים היו בתרבית לבד או יחד עם MSC אדם במגע ישיר על microslides Ibidi או בצדדים מנוגדים של מסנן Transwell למשך 24 שעות. תרבויות היו מגורה עם אלפא גורם נמק גידול (TNFα) למשך 4 שעות ושולבו assay הידבקות מבוסס זרימה. בולוס של נויטרופילים היה perfusedעל האנדותל למשך 4 דקות. לכידתו של זורם נויטרופילים ויחסי הגומלין שלהם עם האנדותל הייתה דמיינה ידי מיקרוסקופ שלב ניגודיות. בשני המודלים, ציטוקינים גירוי מוגבר גיוס האנדותל של נויטרופילים זורמים באופן תלוי מינון. ניתוח של ההתנהגות של נויטרופילים גויסו הראה ירידה תלויה-מינון בגלגול ועלייה תלוי-מינון בגלגול דרך האנדותל. בשיתוף התרבות, MSC מודחק הידבקות נויטרופילים להאנדותל מגורה TNFα. המודלים המבוססת על הידבקות הזרימה שלנו לחקות את השלבים הראשונים של גיוס לויקוציטים מהמחזור. בנוסף לויקוציטים, הם יכולים לשמש כדי לבחון את הגיוס של סוגי תאים אחרים, כגון תאים סרטניים MSC או מחזור מנוהלים טיפולי. דגמי שיתוף התרבות רב-שכבתי שלנו הראו כי MSC לתקשר עם האנדותל לשנות את תגובתם לציטוקינים פרו-דלקתיים, altering הגיוס של נויטרופילים. מחקר נוסף תוך שימוש במודלים אלה נדרש כדי להבין איך תאי סטרומה מרקמות שונות ותנאים (מחלות דלקתיות או סרטן) להשפיע על הגיוס של לויקוציטים במהלך דלקת.

בתחילה, נתחנו את ההשפעה של גירוי EC עם TNFα על הגיוס של נויטרופילים מזרימה באמצעות מודל Ibidi microslide (סעיף 7 - 9). בהעדר TNFα, מעט אם בכלל נויטרופילים דבקו monolayer האנדותל (איור 2 א). זה היה צפוי, שכלא טופל / המנוחה EC אינו מבטא את המולקולות הדרושות הידבקות (selectins) או כ...

Watch this Scientific Journal Video about Analyzing the Effects of Stromal Cells on the Recruitment of Leukocytes from Flow at JoVE.com

Analyzing the Effects of Stromal Cells on the Recruitment of Leukocytes from Flow

היכולת של האנדותל מודלק לגייס leukocytes מזרימה מוסדרת על ידי תאי סטרומה mesenchymal. אנו מתארים שני מודלים במבחנה בשילוב תאים אנושיים עיקריים שיכול לשמש כדי להעריך גיוס נויטרופילים מזרימה ולבחון את התפקיד שתאי סטרומה mesenchymal לשחק בויסות התהליך הזה.

ניתוח ההשפעות של תאי סטרומה על הגיוס של לויקוציטים מתזרים

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we ...

analyzing-effects-stromal-cells-on-recruitment-leukocytes-from

Research

JoVE Journal

Immunology and Infection

10.1K Views.  University of Birmingham.  היכולת של האנדותל מודלק לגייס leukocytes מזרימה מוסדרת על ידי תאי סטרומה mesenchymal. אנו מתארים שני מודלים במבחנה בשילוב תאים אנושיים עיקריים שיכול לשמש כדי להעריך גיוס נויטרופילים מזרימה ולבחון את התפקיד שתאי סטרומה mesenchymal לשחק בויסות התהליך...

Video: Analyzing the Effects of Stromal Cells on the Recruitment of Leukocytes from Flow

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

We describe a method for isolation and characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. Infection of susceptible mouse-strains with this parasite strain results in the induction of experimental cerebral malaria, a neurologic syndrome that recapitulates certain important aspects of Plasmodium falciparum-mediated severe malaria in humans 1,2 . Mature forms of blood-stage malaria express parasitic proteins on the surface of the infected erythrocyte, which allows them to bind to vascular endothelial cells. This process induces obstructions in blood flow, resulting in hypoxia and haemorrhages 3 and also stimulates the recruitment of inflammatory leukocytes to the site of parasite sequestration.
Unlike other infections, i.e neutrotopic viruses4-6, both malaria-parasitized red blood cells (pRBC) as well as associated inflammatory leukocytes remain sequestered within blood vessels rather than infiltrating the brain parenchyma. Thus to avoid contamination of sequestered leukocytes with non-inflammatory circulating cells, extensive intracardial perfusion of infected-mice prior to organ extraction and tissue processing is required in this procedure to remove the blood compartment. After perfusion, brains are harvested and dissected in small pieces. The tissue structure is further disrupted by enzymatic treatment with Collagenase D and DNAse I. The resulting brain homogenate is then centrifuged on a Percoll gradient that allows separation of brain-sequestered leukocytes (BSL) from myelin and other tissue debris. Isolated cells are then washed, counted using a hemocytometer and stained with fluorescent antibodies for subsequent analysis by flow cytometry.
This procedure allows comprehensive phenotypic characterization of inflammatory leukocytes migrating to the brain in response to various stimuli, including stroke as well as viral or parasitic infections. The method also provides a useful tool for assessment of novel anti-inflammatory treatments in pre-clinical animal models.

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

A method for isolation of adherent inflammatory leukocytes from brain blood vessels of Plasmodium berghei ANKA-infected mice is described. The method allows quantification as well as phenotypic characterization of isolated leukocytes after staining with fluorescent antibodies and subsequent analysis by flow cytometry.

בידוד ואנליזה של Leukocytes המוחי מוחרם מ<em&gt; Plasmodium berghei</em&gt; עכברי אנקה נגוע

We describe a method for isolation and  characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. ...

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a prerequisite for pneumococcal infections such as pneumonia and otitis media. In vitro adherence assays can be used to study the attachment of pneumococci to epithelial cell monolayers and to investigate potential interventions, such as the use of probiotics, to inhibit pneumococcal&#160;colonization.&#160;The protocol described here is used to investigate the effects of the probiotic Streptococcus salivarius on the adherence of pneumococci to the human epithelial cell line CCL-23 (sometimes referred to as HEp-2 cells). The assay involves three main steps: 1) preparation of epithelial and bacterial cells, 2) addition of bacteria to epithelial cell monolayers, and 3) detection of adherent pneumococci by viable counts (serial dilution and plating) or quantitative real-time PCR (qPCR). This technique is relatively straightforward and does not require specialized equipment other than a tissue culture setup. The assay can be used to test other probiotic species and/or potential inhibitors of pneumococcal colonization and can be easily modified to address other scientific questions regarding pneumococcal adherence and invasion.

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

In vitro adherence assays can be used to study the attachment of Streptococcus pneumoniae to epithelial cell monolayers and to investigate potential interventions such as the use of probiotics for inhibiting pneumococcal colonization.

חוקר את ההשפעות של פרוביוטיקה על דלקת ריאות התיישבות שימוש<em&gt; במבחנה</em&gt; Assay דבקות

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a ...

Isolation of Leukocytes from the Human Maternal-fetal Interface

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua parietalis). This interface is the anatomical site of contact between maternal and fetal tissues; therefore, it is an immunological site of action during pregnancy. Infiltrating leukocytes at the maternal-fetal interface play a central role in implantation, pregnancy maintenance, and timing of delivery. Therefore, phenotypic and functional characterizations of these leukocytes will provide insight into the mechanisms that lead to&#160;pregnancy disorders. Several protocols have been described in order to isolate infiltrating leukocytes from the decidua basalis and decidua parietalis; however, the lack of consistency in the reagents, enzymes, and times of incubation makes it difficult to compare these results. Described herein is&#160;a novel approach that combines the use of gentle mechanical and enzymatic dissociation techniques to preserve the viability and integrity of extracellular and intracellular markers in leukocytes isolated from the human tissues at the maternal-fetal interface. Aside from immunophenotyping, cell culture, and cell sorting, the future applications of this protocol are numerous and varied. Following this protocol, the isolated leukocytes can be used to determine DNA methylation, expression of target genes, in vitro leukocyte functionality (i.e., phagocytosis, cytotoxicity, T-cell proliferation, and plasticity, etc.), and the production of reactive oxygen species at the maternal-fetal interface. Additionally, using the described protocol, this laboratory has been able to describe new and rare leukocytes at the maternal-fetal interface.

Described herein is a protocol to isolate and further study the infiltrating leukocytes of the decidua basalis and decidua parietalis - the human maternal-fetal interface. This protocol maintains the integrity of cell surface markers and yields enough viable cells for downstream applications as proven by flow cytometry analysis.

בידוד של לויקוציטים מממשק האדם-האימהי העוברי

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua ...

Isolation of Leukocytes from the Murine Tissues at the Maternal-Fetal Interface

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing fetus. This tolerance is initiated during coitus, established during fecundation and implantation, and maintained throughout pregnancy. Active cellular and molecular mediators of maternal-fetal tolerance are enriched at the site of contact between fetal and maternal tissues, known as the maternal-fetal interface, which includes the placenta and the uterine and decidual tissues. This interface is comprised of stromal cells and infiltrating leukocytes, and their abundance and phenotypic characteristics change over the course of pregnancy. Infiltrating leukocytes at the maternal-fetal interface include neutrophils, macrophages, dendritic cells, mast cells, T cells, B cells, NK cells, and NKT cells that together create the local micro-environment that sustains pregnancy. An imbalance among these cells or any inappropriate alteration in their phenotypes is considered a mechanism of disease in pregnancy. Therefore, the study of leukocytes that infiltrate the maternal-fetal interface is essential in order to elucidate the immune mechanisms that lead to pregnancy-related complications. Described herein is a protocol that uses a combination of gentle mechanical dissociation followed by a robust enzymatic disaggregation with a proteolytic and collagenolytic enzymatic cocktail to isolate the infiltrating leukocytes from the murine tissues at the maternal-fetal interface. This protocol allows for the isolation of high numbers of viable leukocytes (&#62;70%) with sufficiently conserved antigenic and functional properties. Isolated leukocytes can then be analyzed by several techniques, including immunophenotyping, cell sorting, imaging, immunoblotting, mRNA expression, cell culture, and in vitro functional assays such as mixed leukocyte reactions, proliferation, or cytotoxicity assays.

Described herein is a protocol to isolate and analyze the infiltrating leukocytes of tissues at the maternal-fetal interface (uterus, decidua, and placenta) of mice. This protocol maintains the integrity of most cell surface markers and yields enough viable cells for downstream applications including flow cytometry analysis.

בידוד של לויקוציטים מרקמות Murine בממשק האם והעובר

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing ...

The Use of Flow Cytometry to Assess the State of Chromatin in T Cells

During a proper immune response, quiescent T cells become activated upon antigen presentation to their antigen-specific T cell receptor. This leads to clonal proliferation of only those T cells that bear a receptor that recognizes the antigen. Chromatin decondensation is a hallmark of T cell activation and is required for T cells to acquire the ability to proliferate after antigen engagement. This change in chromatin condensation can be detected using antibodies raised against histone proteins. These antibodies cannot bind to their epitopes in na&#239;ve T cells as well as they can in activated T cells. We describe how to simultaneously stain T cell-specific surface markers, track viability with a fixable dead cell stain, and measure chromatin status via intracellular staining of Histone H3 proteins. Stained cells are analyzed by flow cytometry and chromatin condensation status is measured as the mean fluorescence intensity (MFI) of the Histone H3 stain. Chromatin decondensation during T cell activation is demonstrated as an increase in the MFI

Flow cytometry can be utilized to assess the state of chromatin within T cells. This protocol allows scientists to interpret evidence of chromatin decondensation during T cell activation demonstrated by an increase in the mean florescence intensity (MFI) of fluorescent Histone H3 antibodies

השימוש בcytometry הזרימה מנת להעריך את המצב של הכרומטין בתאי T

During a proper immune response, quiescent T cells become activated upon antigen presentation to their antigen-specific T cell receptor. This leads to ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

בידוד תזרים Cytometric ניתוח של תאים חיסוניים ממוח עכבר איסכמי

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method enables researchers to better understand the characteristics of a single cell population without the influence of other cells. Compared to other methods of cell enrichment, such as magnetic-activated cell sorting (MCS), FACS is more flexible and accurate for cell separation due to the ability of phenotype detection by flow cytometry. In addition, FACS is usually capable of separating multiple cell populations simultaneously, which improves the efficiency and diversity of experiments. Although FACS has some limitations, it has been broadly used to purify cells for functional studies in both in vitro and in vivo settings. Here we report a protocol using fluorescence-activated cell sorting to isolate a very rare population of immune cells, plasmacytoid dendritic cells (pDC), with high purity from the bone marrow of lupus-prone mice for in vitro functional studies of pDC.

We report a protocol using fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells (pDC) with high purity from the bone marrow of lupus-prone mice for functional studies of pDC.

מיון תא מופעל קרינה עבור טיהור של תאי דנדריטים Plasmacytoid ממח עצם העכבר

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in diameter. In addition to a characterization of forward and sideward scatter properties, it enables the use of fluorescent labeled markers like antibodies to detect respective structures. Using indirect antibody staining, flow cytometry is employed here to quantify birch pollen allergen (precisely Bet v 1)-loaded particles of 0.5 to 10 &#181;m in diameter in inhalable particulate matter (PM10, particle size &#8804;10 &#181;m in diameter). PM10 particles may act as carriers of adsorbed allergens possibly transporting them to the lower respiratory tract, where they could trigger allergic reactions.
So far the allergen content of PM10 has been studied by means of enzyme linked immunosorbent assays (ELISAs) and scanning electron microscopy. ELISA measures the dissolved and not the particle-bound allergen. Compared to scanning electron microscopy, which can visualize allergen-loaded particles, flow cytometry may additionally quantify them. As allergen content of ambient air can deviate from birch pollen count, allergic symptoms might perhaps correlate better with allergen exposure than with pollen count. In conjunction with clinical data, the presented method offers the opportunity to test in future experiments whether allergic reactions to birch pollen antigens are associated with the Bet v 1 allergen content of PM10 particles &#62;0.5 &#181;m.

Here, we present a protocol to quantify allergen-loaded particles by flow cytometry. Ambient particulate matter particles may act as carriers of adsorbed allergens. We show here that flow cytometry, a method widely used to characterize suspended solids &#62;0.5 &#181;m in diameter, can be used to measure these allergen-loaded particles.

ניתוח תזרים Cytometric נ בית נכנס חלקיקים 1 האלרגנים PM10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in ...

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the development and persistence of many health related infections. One of the most well described and studied biofilms in human disease occurs in chronic pulmonary infection of cystic fibrosis patients. When studying biofilms in the context of the host, many factors can impact biofilm formation and development. In order to identify how host factors may affect biofilm formation and development, we used a static chambered coverglass method to grow biofilms in the presence of host-derived factors in the form of sputum supernatants. Bacteria are seeded into chambers and exposed to sputum filtrates. Following 48 hr of growth, biofilms are stained with a commercial biofilm viability kit prior to confocal microscopy and analysis. Following image acquisition, biofilm properties can be assessed using different software platforms. This method allows us to visualize key properties of biofilm growth in presence of different substances including antibiotics.

This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

לדמיין את ההשפעות של כיח על פיתוח biofilm באמצעות מודל תאיים coverglass

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...

Laminar Flow-based Assays to Investigate Leukocyte Recruitment on Cultured Vascular Cells and Adherent Platelets

The recruitment of leukocytes upon injury or inflammation to sites of injury or tissue damage has been investigated during recent decades and has resulted in the concept of the leukocyte adhesion cascade. However, the exact molecular mechanisms involved in leukocyte recruitment have not yet been fully identified. Since leukocyte recruitment remains an important subject in the field of infection, inflammation, and (auto-) immune research, we present a straightforward laminar flow-based assay to study underlying mechanisms of the adhesion, de-adhesion, and transmigration of leukocytes under venous and arterial flow regimes. The in vitro assay can be used to study the molecular mechanisms that underlie the interactions between leukocytes and their cellular partners in different models of vascular inflammation. This protocol describes a laminar flow-based assay using a parallel-flow chamber and an inverted phase contrast microscope connected to a camera to study the interactions of leukocytes and endothelial cells or platelets, which can be visualized and recorded then analyzed offline. Endothelial cells, platelets, or leukocytes can be pretreated with inhibitors or antibodies to determine the role of specific molecules during this process. Shear conditions, i.e. arterial or venous shear stress, can be easily adapted by the viscosity and flow rate of the perfused fluids and the height of the channel.

Leukocytes avidly interact with vascular cells and platelets after vessel wall injury or during inflammation. Here, we describe a straightforward laminar flow-based assay to characterize the molecular mechanisms that underlie the interactions between leukocytes and their cellular partners.

מבחני מבוסס זרימה שכבתית לחקור ליקוציט גיוס על תאים בתרבית וסקולרית, טסיות חסיד

The recruitment of leukocytes upon injury or inflammation to sites of injury or tissue damage has been investigated during recent decades and has resulted ...