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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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.

Burada iltihaplı endoteli tarafından nötrofillerin dolaşımdaki işe okuyan iki in vitro &quot;vasküler&quot; modelleri açıklar. Bu modellerin önemli bir avantajı, in vivo olarak meydana gelen gibi, sırayla lökosit yapışma kademeli olarak her adımda analiz yeteneğidir. Daha önce TNFa ile uyarılan EC 9,29 ile doza bağlı, nötrofil adhezyonunun artış ve transmigrasyonun gözlemledik. Biz de hem modeller lökosit işe üzerindeki stromal hücrelerin etkilerini incelemek iç...

Enflamasyon çözünürlüğü 1,2 izin vermek için, iltihaplı doku sıkı lökosit girişi düzenleme ve çıkış gerektiren mikrobik enfeksiyon veya doku yaralanmasına karşı koruyucu bir cevaptır. Endotel hücreleri (EC), bu hat, kan damarları, dolaşımdaki lökositlerin doku yerleşik stromal hücreler arasındaki çapraz konuşma Bu işlemi 3 koordine için gereklidir. Ancak, lökositlerin kontrolsüz alımı ve etkisiz boşluk kronik inflamatuar hastalıkların 4 gelişimini destekleyen. Sağlıkta ve hastalıkta lökosit işe Mevcut anlayış eksik ve daha sağlam modeller bu süreci analiz etmek gereklidir. sonrası kılcal venüllerin vasküler EC kanın lökosit işe destek mekanizmaları iyi 1,2,5 tarif edilmiştir. Dolaşım halindeki lökositler, özel bir reseptör (örneğin, VCAM-1, E-selektin, P-selektin) tarafından çekildiğiiltihaplı endotel üzerinde yukarı-regüle edilir. Bu geçici etkileşimler lökosit yüzey bağlı kemokin ve lökositler 6- 11 tarafından ifade integrinler aktive lipit kaynaklı mediatörler (kökenli endotel veya stromal ya) ile etkileşim sağlar. Bu da endotel genelinde ve doku 12- 15 içine yapışma ve sürücüler göç dengeler. Doku içinde, lökositler kendi motilite, fonksiyon ve yaşam 16,17 etkileyen stroma türevli ajanlara maruz işe. Büyüyen kanıtlar güçlü sinyaller gelecek için işe alım süreci şartları lökositlerin her aşamasında alınan düşündürmektedir. Ancak, lökosit işe anlayışımız eksik kalır ve çok az doku içindeki bileşenler şekillendirme lökosit hareketi hakkında bilinmektedir. Birmingham biz lökositlerin işe incelemek için in vitro &quot;vasküler&quot; modelleri birkaç geliştirdik9,18,19 akar. Biz şimdi lökosit işe vasküler AK hareket olarak acil regülatörleri kendi yerel mikroçevresinin değişikliklere yanıt anlıyoruz. Özellikle, doku yerleşik stromal hücreler aktif işe 3 rollerini etkilemek için komşu vasküler AT ile söyleşmek kısmen, inflamatuvar yanıtı düzenler. Daha önce çeşitli stromal hücreler, bir doku-spesifik bir şekilde yapışmasını ve lökositlerin geçişini desteklemek için AT yeteneğini modüle göstermiştir, ve bu etkiler, kronik hastalıkların 13,16,20,21 değişmiş da yol açabilir. Böylece, stromal hücreler her inflamatuar yanıt 22 bağlamı tanımlamanızı doku &#39;adres kodları&#39; kurmak. Daha yakın bir zamanda, kemik iliği türevli MSC (BMMSC) potansiyel olarak her iki nötrofillerin alımına bir azalmaya yol açan, sitokinlere EC tepkisini aşağı regüle ve 23 lenfosit olduğunu göstermiştir. mekanizmalar governing işe aydınlatmıştır in vitro ölçüde izole bir tek hücre tipi (örn, AK) veya protein içeren deneyleri kullandık. Ancak bu çalışmalar dikkate almayın lokal doku çevrenin etkileri (örneğin, stromal hücrelerin varlığı) lökositlerin ve doku içine onların sonraki göç. Burada, stromal hücreleri, spesifik olarak mezenkimal kök hücreler içinde iki akış tabanlı yöntemler (MSC) tarif EC 23 ile birlikte kültürlenmiştir. Bu modeller bize akışından lökosit işe desteklemek için özellikle kendi yeteneği endotel tepkiler üzerine stromal hücre etkisini incelemek için izin verir.

<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

Stromal hücreler, komşu endotel hücreleri ile çapraz konuşma ile inflamasyon sırasında, dolaşımdaki lökositlerin işe düzenler. Burada iltihaplı endotel hücreleri tarafından akışından nötrofillerin dolaşımdaki işe okuyan iki in vitro &quot;vasküler&quot; modelleri açıklar. Bu modellerin önemli bir avantajı, in vivo olarak meydana gelen gibi, sırayla lökosit yapışma kademeli olarak her adımda analiz yeteneğidir. Biz de hem modeller lökosit düzenleyen, bu durumda mezenkimal kök hücrelerin (MSC) stromal hücrelerin rolü, çalışma adapte edilebilir açıklanmıştır. Primer endotelyal hücreleri, Ibidi lamlara veya 24 saat süre ile bir Transwell filtrenin ters tarafta doğrudan temas eden insan MSC ile tek başına veya birlikte kültürlenmiştir. Kültürler, 4 saat süre ile, tümör nekroz faktörü alfa (TNFa) ile uyarılır ve bir akım bazlı yapışma deneyinde dahil edilmiştir. Nötrofil bolus serpildi4 dakika için endotel üzerinde. nötrofil ve endotel ile etkileşimlerini akan yakalama faz-kontrast mikroskobu ile görüntülendi. Her iki modelde de, sitokin uyarım, doza bağımlı bir şekilde akan nötrofil endotel işe artmıştır. Işe nötrofillerin davranış analizi haddeleme doza bağımlı azalma, endotelyum içinden geçişe doza bağımlı bir artış göstermiştir. Ko-kültür olarak, MSC TNFa-uyarılmış endotele nötrofil yapışma bastırdı. Bizim akış tabanlı-yapışma modelleri dolaşımdan lökosit işe başlangıç ​​aşamalarını taklit. Lökosit ek olarak, bu tür terapötik olarak uygulandığı, MSC veya dolaşımdaki tümör hücreleri gibi diğer hücre türleri, işe incelemek için kullanılabilir. Çok katmanlı ko-kültür modelleri MSC pro-enflamatuar sitokinlerin tepkilerini değiştirmek için endotel ile iletişim göstermiştir, alterinötrofil işe ng. Bu tür modeller kullanılarak daha fazla araştırma tamamen farklı doku ve koşullar (inflamatuar bozukluklar veya kanser) için nasıl stromal hücreler anlamak enflamasyon sırasında lökositlerin etkilemek için gereklidir.

Başlangıçta, biz Ibidi mikroslayd modeli (- 9 Bölüm 7) kullanılarak akışından nötrofillerin istihdamı TNFa ile AK uyarıcı etkisi analiz. TNFa, herhangi bir nötrofiller endotel tek tabaka yapıştırılır eğer biraz (Şekil 2A) yokluğunda. Bu 25,26 bağlayıcı desteklemek için gerekli yapışma molekülleri (selektinler) veya kemokinleri ifade etmeyen AK dinlenme / işlenmemiş olarak, bekleniyordu. Bunun aksine, sitokin uyarım önemli bir doza bağlı bir ...

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

akışından lökositleri için iltihaplı endotel kabiliyeti mezenkimal stromal hücreler tarafından düzenlenir. Biz akışından nötrofil işe değerlendirmek ve mezenkimal stromal hücreler bu süreci düzenleyen oynadığı rolü incelemek için kullanılabilir birincil insan hücreleri içeren iki in vitro model tarif.

Akış adlı lökositlerin üzerine Stromal hücreler Etkilerinin İncelenmesi

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 Görüntüleme.  University of Birmingham.  akışından lökositleri için iltihaplı endotel kabiliyeti mezenkimal stromal hücreler tarafından düzenlenir. Biz akışından nötrofil işe değerlendirmek ve mezenkimal stromal hücreler bu süreci düzenleyen oynadığı rolü incelemek için kullanılabilir birincil insan hücreleri içeren iki in vitro model tarif. 

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.

Den Beyin sekestre Lökositler İzolasyonu ve Analizi<em&gt; Plasmodium berghei</em&gt; ANKA-enfekte Fare

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

İmmünoloji ve Enfeksiyon

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.

Bir kullanma Pnömokok Kolonizasyon üzerinde Probiyotiklerin Etkileri incelenmesi<em&gt; In Vitro</em&gt; Yapışma Deneyi

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.

İnsan Maternal-Fetal Arabirimi lökosit İzolasyonu

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.

Maternal Fetal Arayüz at Fare Dokular lökosit İzolasyonu

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

Akış Sitometrinin Kullanımı T Hücrelerinde Kromatin Devleti değerlendirmek için

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.

İskemik Fare Beyin gelen Bağışıklık Hücreleri İzolasyon ve Akım Sitometrisi Analizi

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.

Fare kemik iliğinden plazmasitoid dendritik hücreleri Saflaştırılması floresans ile aktive edilen hücre sınıflandırma

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.

PM10 parçacık bağlı Bet v 1 Allergen Akışı Sitometrik Analizi

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.

Bir odacıklı coverglass Modeli Kullanılarak Biyofilm Kalkınma Sputum Etkilerinin görselleştirme

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.

Lökosit işe kültürlü damar hücreleri ve yapışık trombositler üzerinde araştırmak için Laminar akış tabanlı deneyleri

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