Yazarlar onun teknik yardım için Nancy Mora teşekkür etmek istiyorum. Bu çalışma Consejo Nacional de Ciencia y Tecnologia, Meksika, araştırma destekleri 48573-M ve 168098 tarafından hibe IN212308 ve IN205311-2 Direccion General de Asuntos del Kişisel Academico, Universidad Nacional Autonoma de Mexico, Meksika tarafından finanse edildi.

1. İnsan Kanı gelen Nötrofiller İzolasyonu (PMN) <ol><li> Pıhtılaşma önleyici olarak heparin (10 U / ml) ile 20 ml insan kanı kullanın. Kan venopuncture tarafından sağlıklı yetişkin gönüllüler toplanmıştır. UNAM - Tüm deneyler Instituto de INVESTIGACIONES Biomédicas de Biyoetik Komitesi onayı altında yapılmıştır. </li><li> 15 ml konik santrifüj tüpü içine PBS içinde% 6 dekstran T500, 2 ml koyun ve kan 10 ml eklenir. Ters tüp ile iki veya üç kez karıştırın ve eritrosit sedimantasyon için izin vermek için 45 dakika bekletin. </li><li> Taze bir 15 ml konik santrifüj tüpünde 5 ml Ficoll-Paque koydu. </li><li> Sedimanlaşmış eritrositler üzerinde oluştuğunu lökosit zengin plazma alın ve dikkatlice ikinci bir katman oluşturan Ficoll-Paque üstüne Pipeti. Iki faz vardır emin olun. </li><li> 4 ° C&#39;de 20 dakika süre ile 516 x g&#39;de santrifüjleyin Santrifüjlemeden sonra, plazma ve Ficoll-Paque bir ara yüzeyinde mononükleer hücrelerin bir tabakası vardır. Neutrophils tüpün alt tarafında yer alır. </li><li> Süpernatant ortadan kaldırır, bir raf karşı tüp dokunarak hücre peleti kırmak ve 10 ml soğuk (4 ° C) PBS Not eklenerek hücrelerin tekrar süspansiyon:. Yukarı pipetleme hücreleri resuspending ve bu da olduğu aşağı tavsiye edilmez hücrelere zarar. </li><li> 4 ° C&#39;de 5 dakika boyunca 290 xg&#39;de 50 ml konik santrifüj tüpü ve santrifüj hücre süspansiyonu aktarın </li><li> Daha önce olduğu gibi hücre peleti kırmak ve 10 mL soğuk (4 ° C), eritrositlerin lyse hipotonik çözelti (% 0.2 NaCl,% 1 BSA, 20 mM Hepes, pH = 7.4) ekleyin. Tam bir dakika boyunca elle tüpü yavaşça dönen çevirerek karıştırın. </li><li> 10 ml soğuk (4 ° C) hipertonik çözelti (% 1.6 NaCl,% 1 BSA, 20 mM Hepes, pH değeri = 7.4) karışımı ve bir hemositometre kullanılarak hücrelerin sayısı (genellikle&gt;% 95 PMN vardır). Ekleme Not: hücrelerin sayılması sırasında buz üzerinde hücre süspansiyonu ile tüp tutun. </li><li> Daha önce olduğu gibi santrifüjden sonra tekrar süspansiyon 10 PMNSoğuk 7 hücre / ml (4 ° C) PBS ile yıkandı. Buz üzerinde tutun. Not: Nötrofiller 4 de yaşatılabilir ve işlevsel kalır yaklaşık 6 ila 8 saat boyunca ° C. </li></ol> 2. PMN etkinleştirilmesi <ol><li> 100 PMN süspansiyon ul (1 x 10 7 hücre / ml) 1.5 ml Eppendorf tüp içine koymalısınız. Not: Örnek tüpleri en azından çiftleri halinde yapılmalıdır. Olağan negatif kontroller yalnızca ilk antikor ve sekonder antikor içermelidir. </li><li> 10 ug / ml &#39;de karşılık gelen anti-integrin ya da anti-Fc reseptörü monoklonal antikor (mAb) ekleyin ve 15 dakika süreyle buz üzerinde inkübe edilir. </li><li> 1 ml soğuk (4 ° C) PBS, bir mikrosantrifüj içinde 1,743 x g&#39;de (4500 rpm) santrifüje tabi tutarak, ve süpernatan aspire. Eklenmesi ile yıkayın PMN </li><li> Bir raf karşı tüpün alt dokunarak hücre peleti Break, 1 ml soğuk PBS ekleyin ve iki kez daha, önceki adımda olduğu gibi yıkayın. Not: Bu ilişkisiz antikor kaldırmak yıkar. </li><li> Aynı süspanse PMN (inılık itial) hacim (37 ° C) PBS F 60 ug / ml (ab &#39;) 2 keçi anti-fare IgG içeren. Not: ikincil anti-fare IgG antikoru ilk anti-reseptör antikor bağlanacaktır ve reseptör ile çapraz neden olur. </li><li> İlgi nükleer faktör bağlı olarak 1 ila 20 dakika, 37 ° C&#39;de inkübe edin. NF-KB, genellikle 15 dakika için, ve genellikle Elk-1 5 dak. Not: 37 hücreleri inkübe 4 yer alamaz ° C baskılanması reseptör çapraz, ° C </li><li> 1 ml soğuk PBS ekleyin ve mikrosantrifüj yılında 1.743 x g&#39;de 3 dakika santrifüj. </li><li> Süpernatantı </li><li> Dry-ice/ethanol banyosunda hemen PMN ve 10 dakika boyunca onları orada tutmak dondurun. </li></ol> 3. Çekirdeğin İzolasyon ve Fiksasyon <ol><li> 100 ul soğuk içinde süspanse dondurulmuş PMN pellet (4 ° C), hipotonik tampon maddesi (10 mM HEPES, 10 mM KCl, 1.5 mM MgCl2, ve 1 mM taze katma DL-ditiyotretol [DTT]; PH = 7.9). Bu bir tarafından dry-ice/ethanol banyo biri tüp çıkarmak için, ve hipotonik tampon eklemeden önce tüpün alt silin tavsiye edilir. </li><li> Buz üzerine yerleştirin ve tripan mavisi ile çekirdeklerin süspansiyonu bir kısım boyanarak çekirdeklerin bütünlüğünü kontrol edin. Çekirdekler yuvarlak ve mavi görünüyor. Bozulmamış PMN lekeli ve hücre artıkları Düzensiz şekilli mavi parçacıklar olarak görünür alamadım. Not: çekirdekler askı birçok sağlam hücreler veya enkaz içeriyorsa, hazırlık atmak ve başka bir örneği ile işlemi tekrarlayın daha iyidir. </li><li> Soğuk bir oda içinde 10 dakika süreyle mikrosantrifüj 775 xg (3.000 rpm) çekirdekler santrifüjleyin. Bu noktada çekirdekler anda çok kırılgan ve ekstra bakım örnekler soğuk ve aşırı güçler santrifüj değil tutmak alınmalıdır vardır. </li><li> Çok dikkatli pipetleme tarafından süpernatant çıkarın. </li><li> Çekirdekler düzeltmek için PBS içinde soğuk% 4 paraformaldehid 100 ul ekleyin. Not: pelet çok bir gevşek bird arabellek ekleyerek çekirdekleri tekrar süspansiyon için yeterlidir. Aşağı yukarı pipetleme ve kaçınılmalıdır. </li><li> 20 dakika boyunca buz üzerinde inkübe edin. </li><li> Immunolabel Akım sitometri için çekirdekleri veya 4 yukarı 24 saat için onları tutmak ° C </li></ol> 4. Akım Sitometri Analiz Çekirdekler immün <ol><li> 1 dakika mikrosantrifüj yılında 1,743 xg (4.500 rpm) nükleuslu santrifüjleyin ve çok nazik pipetleme tarafından süpernatant kaldırmak. </li><li> 100 ul soğuk (4 ° C)% 0.1 Triton X-100, çekirdeğin geçirgenliği PBS içinde% 4 paraformaldehid. Ekleme 10 dakika buz içinde inkübe edin. </li><li> Santrifüj mikrosantrifüj yılında 1.743 xg&#39;de nükleuslu permeabilize ve dikkatlice süpernatant kaldırmak. Bu noktada çekirdekler granül tüpün alt kısmında da binmemelidir. Bu pelet gevşek alırsa tekrar santrifüj önerilir. </li><li> Spesifik olmayan bağlanma yerleri blok, ve 20 dakika süreyle buz üzerinde inkübe soğuk PBS içinde% 4 fetal bovin serumu (FBS), 500 ul içinde süspanse çekirdekleri. Santrifüj1.743 at çekirdekler mikrosantrifüj 3 dakika xg ve dikkatlice süpernatant kaldırmak. </li><li> 4% FBS ve 2.5 mikrogram / ilgi nükleer faktör karşı mAb ml içeren soğuk PBS 100 ul çekirdekleri tekrar. 20 dakika boyunca buz üzerinde inkübe edin. Not: Her zamanki negatif kontrolleri yalnızca anti-nükleer faktör antikoru içerir ve FITC-etiketli sekonder antikoru sadece olmalıdır. </li><li> 4% FBS ve 3 dk süreyle 1743 xg&#39;de santrifüj ile soğuk PBS 500 ul ile iki kez yıkayın. </li><li> % 4 FBS ve 10 ug / karşılık gelen ikincil FITC-etiketlenmiş antikor ml&#39;lik soğuk PBS içinde 100 ul, çok dikkatli bir şekilde tekrar süspansiyon çekirdekler süpernatantı, ve 20 dakika süreyle buz üzerinde inkübe edilir. </li><li> Önceki aşamada olduğu gibi% 4 FBS ile soğuk PBS 500 ul ile iki kez çekirdekler yıkayın. </li><li> PBS içinde soğuk% 4 paraformaldehid 400 ul çekirdekleri tekrar. Not: Çekirdek numune kontaminasyonu olmaksızın birkaç gün boyunca saklanır izin verecek şekilde yerleştirilir paraformaldehitserum varlığında meydana gelen sorunlar. </li><li> Üç gün için 4 de flow sitometri veya karanlıkta mağaza ° C tarafından hemen analiz edin. </li></ol> 5. Akış Sitometri Analizi <ol><li> Veya benzeri cihazlar; akış sitometresinin böyle bir FACScan (Franklin Lakes, NJ Becton Dickinson) olarak immunolabeled çekirdekleri analiz edin. </li><li> 196 de log-ölçekte SSC ve bir nokta-arsa kapısı çekirdekleri, 10-1 de log-ölçekte FSC:, satın alma ayarları yapın. </li><li> Numune başına on bin çekirdekler edinin. </li><li> 400 log-ölçekte belirlenen FL-1 kanal (506 nm) ile FITC-lekeli çekirdeklerin floresans analiz. </li></ol>

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Yazarlar hiçbir rakip mali çıkarlarının olmadığını beyan ederim.

Burada anlatılan arınma yöntemi kısa bir süre içinde% 95 (mikroskobik gözlem yoluyla belirlenmiştir), daha yüksek saflıkta uyarılmamış nötrofillerin izolasyonu (PMN) verir. İkincisi tamamen lize değilseniz Bazen nötrofiller eritrositler kirlenmiş olabilir. Eritrositler ve PMN kolayca flow sitometri ile farklı hücre popülasyonlarının olarak ayırt edilebilir çünkü bu, genellikle, teknik etkilemez. İzole PMN sonra spesifik monoklonal antikorlar ile çapraz bağlama belirli reseptörler ile stim?...

Nötrofiller, periferik kan 1. en bol bulunan lökositlerin vardır. Inflamasyon ve enfeksiyon nötrofiller sırasında onlar savunma 2 ilk satırı olarak hareket nereye etkilenen sitesinde görünmeye ilk hücrelerdir. Nötrofiller fagositoz, reaktif oksijen türlerinin üretimi, degranülasyonu tarafından parçalayıcı enzimlerin salınımı, ve 4,5 proinflamatuar sitokinlerin üretimi dahil olmak üzere birçok antimikrobiyal mekanizmaları 3 sahiptirler. Nötrofiller hızla çeşitli hücre yüzey reseptörler aracılığıyla aktif olsun kısa ömürlü hücrelerdir. Nötrofiller, kısa ömrü nedeniyle terminali hücreler sayılır ve inflamatuar süreç 6 sırasında aktive sürece apoptoz geçmesi, çünkü onlar da belirli genlerin transkripsiyon düzeyini değiştirerek onların fenotip değiştirebilirsiniz artık açıktır olmasına rağmen. Sitokinler 5 ve apoptoz ile 7,8 inhibisyonu üretim iki tanesi important aktivasyonu-bağımlı hücre fonksiyonlarını nötrofillerde transkripsiyon seviyesinde düzenlenir. Nükleer faktör kappa B (NF-kB) sitokin üretimi 4 transkripsiyonel kontrolü ve hücre canlılığı ve çeşitli hücre tiplerinde apoptoz 9-11 düzenlenmesinde katılır. Nükleer faktör aktivasyonu neden sinyal yollarının genellikle muhabir gen deneyleri veya elektroforetik hareketlilik kayma analizi (EMSA) tarafından incelenmiştir. Nötrofiller kısa ömürlü hücreler, çünkü nötrofil transfeksiyon için bir etkili teknikler vardır Bununla beraber, bu hücreler içinde transkripsiyonel yanıtlar düzenlenmiş çalışması, haberci gen deneyleri ile gerçekleştirilemez. EMSA deneyleri nükleer faktör aktivasyon 12,13 keşfetmek için nötrofillerin kullanılmış olan, bu radyoaktif maddelerin kullanımı içerir çünkü ancak bu yöntem karmaşık ve pahalıdır. Nucleofection successfu kullanılmış olan diğer bir tekniktirlly monositler 14 transferinde. Böylece, en azından teoride, nükleer faktör aktivasyonu (düşük verimlilik rağmen) transfeksiyon tarafından nötrofil tespit edilebilir. Bununla birlikte, bu yöntem daha pahalı ve zaman alıcı bir olasılıkla daha az miktar olacaktır. Immunohistokimyasal hücrelerin mikroskopik analiz aynı zamanda çekirdek içinde nükleer faktör tespit etmek için kullanılabilir. Nitekim, biz çekirdeği bu şekilde 15 içine NF-KB translokasyon tespit ettik. Ne yazık ki, bu tekniğin ayrıca, zaman alıcı az nicel ve gözlemcinin önyargı tabidir. Burada, flow sitometri ile izole ve immunolabeled çekirdeklerindeki nükleer faktörlerin tespiti ve ölçümü sağlayan basit ve etkili bir yöntem sunuyoruz. Biz insan periferik kan nötrofillerin izole teknikleri tanımlamak, (F, anti-reseptör antikorları ile integrinler veya Fc reseptörleri aracılığıyla bu hücreleri uyarmak yalıtmak ve immunolabel çekirdekler ve flow sitometri ile çekirdekler analizŞEKIL 1). Yöntem başarılı bir NF-KB 15 ve nötrofil çekirdekler Elk-1 16 nükleer faktör tespit etmek için kullanılmıştır. Bu yöntemin duyarlılığı çekirdek içinde nükleer faktör seviyeleri küçük değişiklikleri algılama izin verir. Bu yöntem ayrıca diğer hücre tipleri arasından çekirdeklerinde transkripsiyon faktörlerinin düzeyini analiz etmek için de kullanılabilir.

<table border="1">
<tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>REAGENTS</td>
 </tr>
 <tr>
 <td >Heparin </td>
 <td >PiSA (Mexico)</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td >Dextran T500</td>
 <td >Pharmacosmos A/S (Holbaek, Denmark) </td>
 <td>T1-Dextran T500</td>
 <td></td>
 </tr>
 <tr>
 <td >Ficoll-Paque</td>
 <td >Pharmacia</td>
 <td>17-0320-01 </td>
 <td></td>
 </tr>
 <tr>
 <td >Sodium chloride </td>
 <td >Sigma</td>
 <td>S7653 </td>
 <td></td>
 </tr>
 <tr>
 <td >Sodium phosphate monobasic</td>
 <td >Sigma</td>
 <td>S9638</td>
 <td></td>
 </tr>
 <tr>
 <td >Sodium phosphate dibasic</td>
 <td >Sigma</td>
 <td>S9390</td>
 <td></td>
 </tr>
 <tr>
 <td >Bovine serum albumin (BSA)</td>
 <td >Sigma</td>
 <td>A2153</td>
 <td>Cohn Fraction V</td>
 </tr>
 <tr>
 <td >HEPES</td>
 <td >Sigma</td>
 <td>H3375 </td>
 <td></td>
 </tr>
 <tr>
 <td >Potassium chloride</td>
 <td >Sigma</td>
 <td>P9541 </td>
 <td></td>
 </tr>
 <tr>
 <td >Magnesium chloride anhydrous </td>
 <td >Sigma</td>
 <td>M8266 </td>
 <td></td>
 </tr>
 <tr>
 <td >DL-dithiothreitol (DTT) </td>
 <td >Sigma</td>
 <td>D9163</td>
 <td></td>
 </tr>
 <tr>
 <td >Trypan Blue (0.4 % solution)</td>
 <td >Sigma</td>
 <td>T8154</td>
 <td></td>
 </tr>
 <tr>
 <td >Paraformaldehyde</td>
 <td >Sigma</td>
 <td>P6148 </td>
 <td></td>
 </tr>
 <tr>
 <td >Triton X-100 </td>
 <td >Sigma</td>
 <td>X100</td>
 <td></td>
 </tr>
 <tr>
 <td >Fetal bovine serum (FBS)</td>
 <td >GIBCO</td>
 <td>10437-028</td>
 <td></td>
 </tr>
 <tr>
 <td >Monoclonal antibody IV.3</td>
 <td >Medarex (Annandale, NJ) </td>
 <td>025-1 </td>
 <td>Human-specific anti-FcRII (CD32) </td>
 </tr>
 <tr>
 <td >Monoclonal antibody 3G8</td>
 <td >Medarex (Annandale, NJ) </td>
 <td>028-2 </td>
 <td>Human-specific anti-FcRIII (CD16) </td>
 </tr>
 <tr>
 <td >Monoclonal antibody TS2/16</td>
 <td >Dana Farber Cancer Research Institute (Boston, MA) </td>
 <td>Donated by Dr. Martin Hemler </td>
 <td>Human-specific anti-&beta;1 integrin (CD29)</td>
 </tr>
 <tr>
 <td >Monoclonal antibody IB4</td>
 <td >University of California, San Francisco </td>
 <td>Donated by Dr. Eric J. Brown </td>
 <td>Human-specific anti-&beta;2 integrin (CD18)</td>
 </tr>
 <tr>
 <td >F(ab')2 goat anti-mouse IgG </td>
 <td >Cappel (Aurora, OH)</td>
 <td>55468</td>
 <td></td>
 </tr>
 <tr>
 <td>FITC-conjugated F(ab')2 goat anti-mouse IgG</td>
 <td >Cappel (Aurora, OH)</td>
 <td>55522</td>
 <td></td>
 </tr>
 <tr>
 <td>FITC-conjugated F(ab')2 goat anti-rabbit IgG</td>
 <td >Cappel (Aurora, OH)</td>
 <td>55665</td>
 <td></td>
 </tr>
 <tr>
 <td>Anti-NF-&kappa;B p50</td>
 <td >Santa Cruz Biotechnology (Santa Cruz, CA)</td>
 <td>sc-114</td>
 <td>Rabbit polyclonal antibody</td>
 </tr>
 <tr>
 <td>Anti-NF-&kappa;B p65</td>
 <td >Santa Cruz Biotechnology (Santa Cruz, CA)</td>
 <td>sc-109</td>
 <td>Rabbit polyclonal antibody</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>EQUIPMENT</td>
 </tr>
 <tr>
 <td>15-ml centrifuge tube</td>
 <td >Corning</td>
 <td>430791</td>
 <td></td>
 </tr>
 <tr>
 <td>50-ml centrifuge tube</td>
 <td >Corning</td>
 <td>430291</td>
 <td></td>
 </tr>
 <tr>
 <td>Centrifuge, Sorvall Tabletop</td>
 <td >Dupont Instruments</td>
 <td>RT 6000D</td>
 <td></td>
 </tr>
 <tr>
 <td>pH-meter</td>
 <td >Corning</td>
 <td>340</td>
 <td></td>
 </tr>
 <tr>
 <td>Pipetman pipette P-20</td>
 <td >Gilson </td>
 <td>F123600</td>
 <td></td>
 </tr>
 <tr>
 <td>Pipetman pipette P-200</td>
 <td >Gilson </td>
 <td>F123601</td>
 <td></td>
 </tr>
 <tr>
 <td>Pipetman pipette P-1000</td>
 <td >Gilson </td>
 <td>F123602</td>
 <td></td>
 </tr>
 <tr>
 <td>Hemocytometer</td>
 <td >Fisher Scientific</td>
 <td>0267110</td>
 <td></td>
 </tr>
 <tr>
 <td>Microscope</td>
 <td >Nikon </td>
 <td>Eclipse E600</td>
 <td></td>
 </tr>
 <tr>
 <td>Inverted microscope</td>
 <td >Nikon </td>
 <td>TMS</td>
 <td></td>
 </tr>
 <tr>
 <td>Water Bath Incubator</td>
 <td >Fisher Scientific </td>
 <td>2IS-M</td>
 <td></td>
 </tr>
 <tr>
 <td>Microcentrifuge</td>
 <td >Eppendorf</td>
 <td>5414C</td>
 <td></td>
 </tr>
 <tr>
 <td>Microcentrifuge</td>
 <td >Eppendorf</td>
 <td>5418</td>
 <td></td>
 </tr>
 <tr>
 <td>Flow Cytometer</td>
 <td >Becton Dickinson (Franklin Lakes, NJ) </td>
 <td>FACScalibur </td>
 <td></td>
 </tr>
 </tbody>
</table>

a simple and efficient method to detect nuclear factor activation in human neutrophils by flow cytometry

Nötrofiller periferik kanda en bol lökosit vardır. Bu hücreler böylece mikroorganizmaların işgalci karşı ilk savunma hattı olma, inflamasyon ve enfeksiyon sitelerinde görünmesini ilk. Nötrofiller gibi fagositoz, parçalayıcı enzimlerin salınımı, ve reaktif oksijen türlerinin üretimi gibi önemli antimikrobiyal fonksiyonları sahip. Bu önemli savunma işlevlerine ek olarak, nötrofillerin proinflamatuar sitokinlerin ve apopitoz inhibisyonu üretimi gibi enfeksiyonuna yanıt olarak diğer görevleri yerine getirir. Sitokinler enfeksiyonu temizlemek yardımcı olan diğer lökositler işe ve apopitoz inhibisyonu nötrofil enfeksiyon yerinde uzun yaşamalarını sağlar. Bu işlevler, transkripsiyon seviyesinde düzenlenir. Nötrofiller kısa ömürlü hücreler çünkü herhangi bir verimli olduğu için Bununla birlikte, bu hücreler içinde transkripsiyonel yanıtlar düzenlenmiş bir çalışma geleneksel raportör gen yöntemler ile gerçekleştirilemezNötrofil transfeksiyon için cient teknikleri. Burada, flow sitometri ile izole ve immunolabeled çekirdeklerindeki nükleer faktörlerin tespiti ve ölçümü sağlayan basit ve etkili bir yöntem sunuyoruz. Biz insan periferik kan saf nötrofilleri izole teknikleri tanımlamak, anti-reseptör antikorları ile bu hücreleri uyarmak yalıtmak ve immunolabel çekirdekler ve flow sitometri ile çekirdekler analiz. Yöntem başarılı bir şekilde nötrofiller ve diğer hücre türleri çekirdeklerinde NF-KB ve Elk-1 nükleer faktör tespit etmek için kullanılmıştır. Bu yüzden, bu yöntem, hücre tiplerinde izole edilen çekirdeklerin içinde transkripsiyon faktörlerinin aktivasyonu analiz etmek için bir seçenek oluşturmaktadır.

Saflaştırma yöntemi burada açıklanan genellikle% 95 (Şekil 1A) daha büyük saflık ile uyarılmamış nötrofiller (PMN) sağlar. İzole PMN sonra spesifik monoklonal antikorlar ile çapraz bağlama belirli reseptörler ile stimüle edilebilir. Biz Fc reseptörleri ve integrinler (Şekil 1B) ile PMN canlandırmıştır. Bir kez uyarılan PMN lize ve çekirdekler yüksek verimle izole edilmektedir. Çekirdekler sonra bu özel antikorlar ile nükleer faktör kappa B (NF-KB) <strong...

Watch this Scientific Journal Video about A Simple and Efficient Method to Detect Nuclear Factor Activation in Human Neutrophils by Flow Cytometry at JoVE.com

A Simple and Efficient Method to Detect Nuclear Factor Activation in Human Neutrophils by Flow Cytometry

Nötrofiller, kan içinde en bol bulunan lökositlerin vardır. Nötrofiller proinflamatuar sitokinlerin ve apopitoz inhibisyonu üretimi gibi transkripsiyonel düzenlenir fonksiyonları sahip. Bu işlevler izole çekirdeklerinde flow sitometri ile nükleer faktörlerin tespiti ve ölçümü sağlar Burada sunulan yöntem ile ele alınabilir

Akım Sitometri tarafından İnsan Nötrofiller Nükleer Faktör Aktivasyon Algılama Basit ve Verimli Yöntemi

Neutrophils  are the most abundant leukocytes in peripheral blood. These cells are the first  to appear at sites of inflammation and infection, thus ...

a-simple-efficient-method-to-detect-nuclear-factor-activation-human

Research

JoVE Journal

Immunology and Infection

17.7K Görüntüleme.  University of Alberta. Nötrofiller, kan içinde en bol bulunan lökositlerin vardır. Nötrofiller proinflamatuar sitokinlerin ve apopitoz inhibisyonu üretimi gibi transkripsiyonel düzenlenir fonksiyonları sahip. Bu işlevler izole çekirdeklerinde flow sitometri ile nükleer faktörlerin tespiti ve ölçümü sağlar Burada sunulan yöntem ile ele alınabilir

Video: A Simple and Efficient Method to Detect Nuclear Factor Activation in Human Neutrophils by Flow Cytometry

Quantitative Assessment of Immune Cells in the Injured Spinal Cord Tissue by Flow Cytometry:  a Novel Use for a Cell Purification Method

Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true quantitative analysis of cellular inflammation. Flow cytometry is a quick alternative method to quantify immune cells in the injured brain or spinal cord tissue. Historically, flow cytometry has been used to quantify immune cells collected from blood or dissociated spleen or thymus, and only a few studies have attempted to quantify immune cells in the injured spinal cord by flow cytometry using fresh dissociated cord tissue. However, the dissociated spinal cord tissue is concentrated with myelin debris that can be mistaken for cells and reduce cell count reliability obtained by the flow cytometer. We have advanced a cell preparation method using the OptiPrep gradient system to effectively separate lipid/myelin debris from cells, providing sensitive and reliable quantifications of cellular inflammation in the injured spinal cord by flow cytometry. As described in our recent study (Beck &amp; Nguyen et al., Brain. 2010 Feb; 133 (Pt 2): 433-47), the OptiPrep cell preparation had increased sensitivity to detect cellular inflammation in the injured spinal cord, with counts of specific cell types correlating with injury severity. Critically, novel usage of this method provided the first characterization of acute and chronic cellular inflammation after SCI to include a complete time course for polymorphonuclear leukocytes (PMNs, neutrophils), macrophages/microglia, and T-cells over a period ranging from 2 hours to 180 days post-injury (dpi), identifying a surprising novel second phase of cellular inflammation. Thorough characterization of cellular inflammation using this method may provide a better understanding of neuroinflammation in the injured CNS, and reveal an important multiphasic component of neuroinflammation that may be critical for the design and implementation of rational therapeutic treatment strategies, including both cell-based and pharmacological interventions for SCI.

Quantification of cellular inflammation in the injured/pathological CNS by flow cytometry is complicated by lipid/myelin debris that can have similar size and granulation to cells, decreasing sensitivity/accuracy. We have advanced a cell preparation method to remove myelin debris and improve cell detection by flow cytometry in the injured spinal cord.

Akış Sitometri Yaralı Omurilik Doku Bağışıklık Hücreleri Kantitatif Değerlendirilmesi: Hücre Arıtma Yöntemi Roman kullanın

Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true ...

İmmünoloji ve Enfeksiyon

A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although the isolation methods of liver macrophages have been well-described4-6, most of these methods require sophisticated equipment, such as a centrifugal elutriator and technical skills. Here, we provide a novel method to obtain liver macrophages in sufficient number and purity from mixed primary cultures of adult rat liver cells, as schematically illustrated in Figure 1.
After dissociation of the liver cells by two-step perfusion method7,8,a fraction mostly composed of parenchymal hepatocytes is prepared and seeded into T75 tissue culture flasks with culture medium composed of DMEM and 10% FCS.Parenchymal hepatocytes lose the epithelial cell morphology within a few days in culture, degenerate or transform into fibroblast-like cells (Figure 2). As the culture proceeds, around day 6, phase contrast-bright, round macrophage-like cells start to proliferate on the fibroblastic cell sheet (Figure 2). The growth of the macrophage-like cells continue and reach to maximum levels around day 12, covering the cell sheet on the flask surface. By shaking of the culture flasks, macrophages are readily suspended into the culture medium. Subsequent transfer and short incubation in plastic dishes result in selective adhesion of macrophages(Figure 3), where as other contaminating cells remain suspended. After several rinses with PBS, attached macrophages are harvested. More than 106 cells can be harvested repeatedly from the same T75 tissue culture flask at two to three day intervals for more than two weeks(Figure 3).The purities of the isolated macrophages were 95 to 99%, as evaluated by flow cytometry or immunocytochemistry with rat macrophage-specific antibodies (Figure 4).The isolated cells show active phagocytosis of polystylene beads (Figure 5), proliferative response to recombinant GM-CSF, secretion of inflammatory/anti-inflammatory cytokines upon stimulation with LPS, and formation of multinucleated giant cells9.
In conclusion, we provide a simple and efficient method to obtain liver macrophages in sufficient number and purity without complex equipment and skills.This method might be applicable to other mammalian species.

A novel method to obtain macrophages from primary culture of rat liver cells is described. This method utilizes the proliferation of macrophages in the culture, followed by shaking of culture flasks and purification by selective attachment to plastic dishes. This technique efficiently provides liver macrophages without complex equipment and skills.

Yetişkin Karaciğer Hücrelerinin Karma İlköğretim Kültürler Makrofajlar yalıtın Basit ve Etkin Yöntem

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although ...

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response following an infection. Despite efforts to identify unique surface marker on Tregs, the only unique feature is their ability to suppress the proliferation and function of effector T cells. While it is clear that only in vitro assays can be used in assessing human Treg function, this becomes problematic when assessing the results from cross-sectional studies where healthy cells and cells isolated from subjects with autoimmune diseases (like Type 1 Diabetes-T1D) need to be compared. There is a great variability among laboratories in the number and type of responder T cells, nature and strength of stimulation, Treg:responder ratios and the number and type of antigen-presenting cells (APC) used in human in vitro suppression assays. This variability makes comparison between studies measuring Treg function difficult. The Treg field needs a standardized suppression assay that will work well with both healthy subjects and those with autoimmune diseases. We have developed an in vitro suppression assay that shows very little intra-assay variability in the stimulation of T cells isolated from healthy volunteers compared to subjects with underlying autoimmune destruction of pancreatic &beta;-cells. The main goal of this piece is to describe an in vitro human suppression assay that allows comparison between different subject groups. Additionally, this assay has the potential to delineate a small loss in nTreg function and anticipate further loss in the future, thus identifying subjects who could benefit from preventive immunomodulatory therapy1. Below, we provide thorough description of the steps involved in this procedure. We hope to contribute to the standardization of the in vitro suppression assay used to measure Treg function. In addition, we offer this assay as a tool to recognize an early state of immune imbalance and a potential functional biomarker for T1D.

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Tregs are potent suppressors of the immune system. There is a lack of unique surface markers to define them, hence, definitions of Tregs are primarily functional. Here we describe an optimized in vitro assay capable of identifying immune imbalance in subjects at risk to develop T1D.

Insan In Vitro Bağışıklık Dengesizlik Erken Devlet Tanıma Tarama Aracı Olarak Bastırılması

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response ...

Examination of Thymic Positive and Negative Selection by Flow Cytometry

A healthy immune system requires that T cells respond to foreign antigens while remaining tolerant to self-antigens. Random rearrangement of the T cell receptor (TCR) &alpha; and &beta; loci generates a T cell repertoire with vast diversity in antigen specificity, both to self and foreign. Selection of the repertoire during development in the thymus is critical for generating safe and useful T cells. Defects in thymic selection contribute to the development of autoimmune and immunodeficiency disorders1-4. 
T cell progenitors enter the thymus as double negative (DN) thymocytes that do not express CD4 or CD8 co-receptors. Expression of the &alpha;&beta;TCR and both co-receptors occurs at the double positive (DP) stage. Interaction of the &alpha;&beta;TCR with self-peptide-MHC (pMHC) presented by thymic cells determines the fate of the DP thymocyte. High affinity interactions lead to negative selection and elimination of self-reactive thymocytes. Low affinity interactions result in positive selection and development of CD4 or CD8 single positive (SP) T cells capable of recognizing foreign antigens presented by self-MHC5.
Positive selection can be studied in mice with a polyclonal (wildtype) TCR repertoire by observing the generation of mature T cells. However, they are not ideal for the study of negative selection, which involves deletion of small antigen-specific populations. Many model systems have been used to study negative selection but vary in their ability to recapitulate physiological events6. For example, in vitro stimulation of thymocytes lacks the thymic environment that is intimately involved in selection, while administration of exogenous antigen can lead to non-specific deletion of thymocytes7-9. Currently, the best tools for studying in vivo negative selection are mice that express a transgenic TCR specific for endogenous self-antigen. However, many classical TCR transgenic models are characterized by premature expression of the transgenic TCR&alpha; chain at the DN stage, resulting in premature negative selection. Our lab has developed the HYcd4 model, in which the transgenic HY TCR&alpha; is conditionally expressed at the DP stage, allowing negative selection to occur during the DP to SP transition as occurs in wildtype mice10.
Here, we describe a flow cytometry-based protocol to examine thymic positive and negative selection in the HYcd4 mouse model. While negative selection in HYcd4 mice is highly physiological, these methods can also be applied to other TCR transgenic models. We will also present general strategies for analyzing positive selection in a polyclonal repertoire applicable to any genetically manipulated mice.

We present a flow cytometry-based method to examine T cell development in vivo using genetically manipulated mice on a wildtype or T cell receptor transgenic background.

Akım Sitometri tarafından Timik Pozitif ve Negatif Seçim incelenmesi

A healthy immune system requires that T cells respond to foreign  antigens while remaining tolerant to self-antigens. Random rearrangement of the  T cell ...

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of phenol soluble modulins (PSMs) and other toxins produced and secreted by Staphylococcus aureus on neutrophils. To study the effects of the PSMs on neutrophils we isolate fresh neutrophils using density gradient centrifugation. These neutrophils are loaded with a dye that fluoresces upon calcium mobilization. The activation of neutrophils by PSMs initiates a rapid and transient increase in the free intracellular calcium concentration. In a flow cytometry experiment this rapid mobilization can be measured by monitoring the fluorescence of a pre-loaded dye that reacts to the increased concentration of free Ca2+. Using this method we can determine the PSM concentration necessary to activate the neutrophil, and measure the effects of specific and general inhibitors of the neutrophil activation.	
	To investigate the expression of the PSMs in the intracellular space, we have constructed reporter fusions of the promoter of the PSM&alpha; operon to GFP. When these reporter strains of S. aureus are phagocytosed by neutrophils, the induction of expression can be observed using fluorescence microscopy.

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of PSMs and other toxins secreted by Staphylococcus aureus on neutrophils using flow cytometry and fluorescence microscopy.

Etkileşimi incelenmesi<em&gt; Staphylococcus aureus</em&gt; Akım Sitometri ve Time Lapse Mikroskopi tarafından Nötrofiller ile

We present methods to study the effect of phenol soluble modulins (PSMs)  and other toxins produced and secreted by Staphylococcus  aureus on neutrophils. ...

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

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted as an important hallmark of cancer. Our research on breast cancer focuses on the active role that tumor infiltrating lymphocytes play in tumor progression and patient outcome. Toward this goal, we developed a methodology for the rapid isolation of intact lymphoid cells from normal and abnormal tissues in an effort to evaluate them proximate to their native state. Homogenates prepared using a mechanical dissociator show both increased viability and cell recovery while preserving surface receptor expression compared to enzyme-digested tissues. Furthermore, enzymatic digestion of the remaining insoluble material did not recover additional CD45+ cells indicating that quantitative and qualitative measurements in the primary homogenate likely genuinely reflect infiltrating subpopulations in the tissue fragment. The lymphoid cells in these homogenates can be easily characterized using immunological (phenotype, proliferation, etc.) or molecular (DNA, RNA and/or protein) approaches. CD45+ cells can also be used for subpopulation purification, in vitro expansion or cryopreservation. An additional benefit of this approach is that the primary tissue supernatant from the homogenates can be used to characterize and compare cytokines, chemokines, immunoglobulins and antigens present in normal and malignant tissues. This protocol functions extremely well for human breast tissues and should be applicable to a wide variety of normal and abnormal tissues.

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.

İnfiltratif Lenfositlerinin Analiz Olmayan enzimatik ayırmak Taze İnsan Dokusuna Basit ve Hızlı Protokolü

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

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.

Basit ve Etkin Yaklaşım Mutant Vaccinia Virüs Vektörler Construct

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

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research investigating immunological responses against A. fumigatus has been limited by the lack of consistent and reliable assays for measuring the antifungal activity of specific immune cells in vitro. A new method is described to assess the antifungal activity of primary monocytes and neutrophils from human donors against A. fumigatus using FLuorescent Aspergillus REporter (FLARE) conidia. These conidia contain a genetically encoded dsRed reporter, which is constitutively expressed by live FLARE conidia, and are externally labeled with Alexa Fluor 633, which is resistant to degradation within the phagolysosome, thus allowing a distinction between live and dead A. fumigatus conidia. Video microscopy and flow cytometry are subsequently used to visualize the interaction between conidia and innate immune cells, assessing fungicidal activity whilst also providing a wealth of information on phagocyte migration, phagocytosis and the inhibition of fungal growth. This novel technique has already provided exciting new insights into the host-pathogen interaction of primary immune cells against A. fumigatus. It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.
It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Here, we describe a protocol to assess antifungal activity of primary human immune cells in real-time using fluorescent Aspergillus reporter conidia in conjunction with live-cell video microscopy and flow cytometry. Generated data provide insight into host cell-Aspergillus interactions such as fungicidal activity, phagocytosis, cell migration and inhibition of fungal growth.

Cevabı İnsan İlköğretim nötrofiller ve monositler tarafından Antifungal Faaliyet Canlı Görüntüleme<em&gt; A. fumigatus</em

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

Yalıtım ve fare ve insan alveoler makrofajlar Vitro kültürü

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can have pathological significance. An ideal method for examining alterations to monocytes is whole blood flow cytometry as the minimal handling of samples by this method limits artifactual cell activation. However, many different approaches are taken to gate the monocyte subsets leading to inconsistent identification of the subsets between studies. Here we demonstrate a method using whole blood flow cytometry to identify and characterize human monocyte subsets (classical, intermediate, and non-classical). We outline how to prepare the blood samples for flow cytometry, gate the subsets (ensure contaminating cells have been removed), and determine monocyte subset expression of surface markers &#8212; in this example M1 and M2 markers. This protocol can be extended to other studies that require a standard gating method for assessing monocyte subset proportions and monocyte subset expression of other functional markers.

Here we present a protocol for characterizing monocyte subsets by whole blood flow cytometry. This includes outlining how to gate the subsets and assess their expression of surface markers and giving an example of the assessment of the expression of M1 (inflammatory) and M2 markers (anti-inflammatory).

İnsan monosit alt kümeleri tam kan tarafından karakterizasyonu Akış Sitometresi Analizi

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can ...