Bu araştırma, Kanada Kan Hizmetleri (CBS) ve Sağlık Bilimleri (CIHR) Araştırma Ortaklığı Fonu Kanada Enstitüleri tarafından finanse edildi. Yazarların kendi araştırma tesislerinin kullanımı için Kan araştırma UBC Merkezi&#39;nde LMB Makromolekül Hub ederim. Altyapı tesisi Yenilik Kanada temeli (CFI) ve Sağlık Araştırma Michael Smith Vakfı (MSFHR) tarafından desteklenmektedir. R. Chapanian (CIHR / CBS) doktora sonrası Transfüzyon Bilim burs ve MSFHR araştırma stajyer doktora sonrası dostluk bir alıcı bir alıcı olduğunu. JN Kizhakkedathu MSFHR Kariyer Araştırmacı Scholar Ödülü bir alıcı olduğunu.

A. Hyperbranched poligliserol Modifikasyonu (SS-HPG) <ol><li> Sıra bir tabanı yuvarlak bir şişe içerisinde 60 kDa (0,5 g, 0,0083 mol) HPG liyofilize edildi ve 90 C&#39;de vakum altında gece boyunca kurutun ° C. </li><li> Oda sıcaklığına soğutmak şişeye, ve susuz piridin (3 ml) içinde kuru HPG çözülür. </li><li> Karboksil grupları ile HPG üzerinde yaklaşık olarak sekiz hidroksil gruplarının fonksiyonalize, HPG çözeltisine dimetilaminopiridin ve katalitik miktarda (piridin içinde 5 mg / ml çözeltisinin bir damla) ekleyin. Bu karışıma, 10 dakika boyunca akıllı 0.5 ml piridin içinde çözülür damla (0.0067 g, 0,0664 mmol) ve süksinik anhidrit ilave edin. Argon altında oda sıcaklığında gece boyunca karıştırılır. </li><li> 50 ml&#39;lik bir santrifüj tüpüne soğuk aseton (4 ° C) 40 ml karışım çökelti, ve 15 dakika süreyle 27,000 x g bir Beckman J2-MC santrifüjü kullanılarak santrifüj. Süpernatant süzün ve oda sıcaklığında, argon püskürtülerek kalıntı aseton çıkarın. </li><li> Karboksi etkinleştirmek içinsüksinimidil süksinat (SS) grupları ile l gruplan, susuz DMF içindeki 3 ml karboksil fonksiyonalitesine sahip HPG çözülür. HPG çözelti, N-hidroksisüksinimid (0.0077 g, 0,0664 mol) ve N, N&#39;-diizopropilkarbodiimid (0,0084 g, 0,0664 mol) eklenir ve argon altında oda sıcaklığında gece boyunca karıştırılır. </li><li> Soğuk aseton yağış tarafından SS-HPG arındırın. </li><li> Argon püskürtülerek kalıntı aseton çıkarın. </li><li> NMR analizi değiştirilmiş - HPG saflık ve proton (1 H) karboksil ve SS işlevsellik derecesini belirler. </li></ol> B. Tam Kan Toplama ve kırmızı kan hücrelerinin ayrılması (eritrositler) <ol><li> Bir sitrat Vacutainer tüpüne sağlıklı insan donörlerden razı tam kan (% 40 Hematokrit, 3 ml) toplayın. </li><li> 4 dakika boyunca 1.000 xg&#39;de sitrat tüp santrifüjleyin. </li><li> Plazma ve trombosit içeren Süpernatantı ve beyaz kan hücreleri ve platele içeren ara buffy coatPasteur pipeti kullanarak ts. </li><li> 12 ml Falcon santrifüj tüpü içine paketlenmiş eritrositler (% 80 Hematokrit, 1.2 ml) aktarın ve eritrosit yıkamak için PBS tamponu (8 mL, pH = 8.0) ilave edilir. </li><li> Üniform hücre dağılımı elde etmek için ters çevirerek RBCs karıştırın. </li><li> 4 dakika boyunca 1.000 xg&#39;de Falcon tüp santrifüjleyin ve süpernatant kaldırmak. </li><li> 5. ve 6. adımları tekrarlayarak PBS iki kez daha sahip eritrositler yıkayın. </li><li> Sıra 1.6 ml Eppendorf tüpüne (100 ul)-alyuvarlar paketlenmiş ve% 20 Hematokrit, eritrositler elde etmek için PBS tamponu (300 ul, pH = 8.0) ilave edilir. </li></ol> C. HPG eritrositler için Aşılama <ol><li> Hemen adımda A6 aseton içinde değiştirilmiş HPG, yer çökelme sonra bir cam şişeye (1 dram) ve PBS tamponu (300 ul, pH = 8.0) içinde çözülür SS-HPG (150 mg). </li><li> % 20 Hematokrit içine SS-HPG polimer solüsyonu eklenir ve 400 ul 0.5 mM ile 3 mm arasında değişir ve SS-HPG konsantrasyonunun bir nihai hacim elde etmek için eritrositler yıkanır. Örneğin, 0.5 mM ile muamele eritrositler hazırlamak için SS-HPG, yıkanmış eritrosit gelen PBS 36 ul kaldırmak ve SS-HPG polimer çözeltisine 36 ul yerleştirin. </li><li> Girdap nazikçe ve süspansiyon 1 saat boyunca oda sıcaklığında bir orbital çalkalayıcı üzerinde Eppendorf tüpleri. </li><li> 4 dakika boyunca 1.000 xg&#39;de Eppendorf tüpler Santrifüj ve supernatant dışarı Pipeti. </li><li> , RBC yıkama santrifüjden süpernatant kaldırmak PBS 1 ml ekleyin. </li><li> 1 ml serum fizyolojik ekleyin ve iki kat 5. adımda RBCs yıkayın. </li><li> % 20 Hematokrit, bir nihai konsantrasyona kadar paketlenmiş eritrositler 100 ul (% 80 Hematokrit) ile tuzlu su 300 ul ekle. </li></ol> Modifiye RBCs HPG D. Karakterizasyonu I. Kompleman aracılı lizis <ol><li> Sağlıklı insan donörlerden alınan serum BD Vacutainer cam tüp içine tam kanın yaklaşık 8 ml toplamak, ve 30 dakika süreyle oda sıcaklığında pıhtılaşmasına izin kan. </li><li> 10 dakika boyunca 2.000 xg&#39;de tüp santrifüjleyin ve serum yaklaşık 3 ml toplamak. </li><li> Seru Bir mililitrem ısıyla inaktive serum hazırlamak için 30 dakika için 60 ° C sıcaklıkta bir su banyosuna yerleştirildi. </li><li> 20% Hematokrit HPG modifiye eritrositler veya değiştirilmemiş RBCs 60 ul içeren bir Eppendorf tüp içine taze serum veya ısı inaktive serum 60 ul ekleyin. </li><li> 37, 1 saat boyunca inkübe eritrositler ° C. </li><li> Hücre süspansiyonu hemoglobin miktar yer 96 kuyulu plaka içinde üç kopya olarak değiştirilmiş ya da değiştirilmemiş alyuvarlar hücreleri HPG% 20 hematokrit 5.9 ul ölçmek için. Drabkin reaktifi (spectrophotomerically hemoglobin miktarını ölçmek için kullanılan bir reaktif) 294 ul ekle. Ve dışarı pipetleme Drabkin reaktifi ile hücreleri karıştırın. SPECTRA MAX 190 plaka okuyucu kullanarak 540 nm&#39;de hemoglobin cyanoderivative arasında absorbansları ölçülür. </li><li> Üstte yüzen madde içindeki hemoglobin miktarını ölçmek için, 1 dakika için 13.000 x g santrifüj hücreleri. 96 plaka üç nüsha halinde süpernatant Yeri 50 ul. Drabkin reaktifi 250 ul ekleyin. Drabkin reaktifi ile karıştırın hücrelerive dışarı pipetleme. SPECTRA MAX 190 plaka okuyucu kullanarak 540 nm&#39;de hemoglobin cyanoderivative arasında absorbansları ölçülür. </li><li> Örnek olarak üstte yüzen madde içindeki hemoglobin oranı (adım 7) ve toplam hemoglobin (adım 6) 8, 11, 12 gelen erimiş hücrelerinin miktarını ölçmek. </li></ol> II. MTS kartlarını kullanarak majör ve minör antijenlerinin kamuflaj <ol><li> Sıra 50 bir Eppendorf tüp HPG modifiye RBC (10% Hematokrit), 1 dk için 1.000 xg&#39;de santrifüj ul ve süpernatant kaldırmak. </li><li> Hücre pelletini MTS seyreltici 110 ul ekleyin ve dışarı pipetleme hücre süspansiyonu homojenize. </li><li> Her mini jel kolon hücre süspansiyonu 11 ul ekleyin. Ilavesi sırasında jel dokunmaktan kaçının. </li><li> Bir santrifüj kartı yuvasına MTS kartları bulun ve 6 dakika boyunca 156 xg&#39;de santrifüj. </li><li> Yüzey antijenlerinin korunması üretimi esas olarak, mini jel kolon içinde RBC konumu belirlenirr açıklaması. </li></ol> III. Sulu iki faz bölüm ölçümleri <ol><li> Dekstran 500 kDa 13 &#39;e göre (% 5 dekstran 500K,% 4 PEG 8K, 150 mM NaCl, 10 mM fosfat) oluşan / PEG 8 kDa iki faz ayırma sistemi hazırlayın. </li><li> Iki fazlı bir sistem (alt dekstran) elde etmek üzere oda sıcaklığında 10 dakika boyunca 433 x g&#39;de santrifüjleyin. </li><li> Dikkatlice iki aşamadan ayırın. </li><li> Bir etiketli tüp (konkav dipli) içine üst PEG faz kısım 1.0 ml getirerek yavaşça 20% 20 hematokrit HPG modifiye RBC ul ve karıştırın hücreleri ekleyin. </li><li> Daha düşük bir faz ile 0.5 mL hücre içerir, ve hafifçe vurarak yavaşça sistemi karıştırmak üst faz 0.5 ml ilave edilir. </li><li> Ayırmak ve sistemindeki hücrelerin in yerini belirlemek için sistem için ~ 2 dk için tezgah üzerinde yerleştirilir. Eritrosit lokalizasyonu (üst faz PEG, alt dekstran faz ya da her ikisi de) ve moleküler değişiklik derecesi ile işlevHPG ağırlığı. </li></ol> IV. Ozmotik frajilite ölçümleri <ol><li> 0 ila 0.9 (w / v)% arasında değişmektedir ve konsantrasyonu NaCl çözeltisi hazırlayın. </li><li> 1.5 ml Eppendorf tüpleri içine NaCl solüsyonlarının Yeri 0.4 ml. </li><li> NaCl solüsyonlarının içine eritrositler (% 20 hematokrit) 20 ul ekleyin. </li><li> Inversiyon ve 30 dakika boyunca 37 ° C&#39;de su banyosunda koyun tarafından dikkatle Mix hücreleri. </li><li> Inversiyon tarafından dikkatle hücreleri erteleyin. RBC süspansiyonu 50 ul al ve bir küvet içine yerleştirilmiş 1 ml Drabkin reaktifi, eklemek. Ve dışarı pipetleme Drabkin reaktifi ile hücreleri karıştırın. BECKMAN COULTER DU 730 UV / VIS okuyucusu kullanarak 540 nm&#39;de hemoglobin cyanoderivative arasında absorbansları ölçülür. </li><li> Üstte yüzen madde içindeki hemoglobin miktarını ölçmek için, 1 dakika için 1000 x g hücreler santrifüj. Bir küvet içinde Drabkin reaktifi 1 ml süpernatan 200 ul ekle. Ve dışarı pipetleme Drabkin reaktifi ile hücreleri karıştırın. Hemogl ve absorbansı ölçülürBECKMAN COULTER DU 730 UV / VIS okuyucusu kullanarak 540 nm&#39;de Tobin cyanoderivative. </li><li> Hücre süspansiyonu (adım 5) içinde hemoglobin toplam miktarı için üstte yüzen madde içindeki hemoglobin miktar oranı (adım 6) farklı NaCI çözeltisi içinde lize hücrelerinin miktarını hesaplar. </li></ol> V. Flow sitometri ölçümleri - Rh-D Korunması (Rh) antijen <ol><li> 5 kontrol ul ve 25 ul toplam hacim ile% 0.5 BSA ile takviye PBS tamponu ile üç kopya halinde HPG değiştirilmiş eritrositler (% 20 hematokrit) ekleyin. </li><li> Bölüm Biodiagnostics (PA, ABD) satın FITC monoklonal anti-Rh D (RHD) 25 ul ekle. </li><li> Bir su banyosu içinde 37 ° C sıcaklıkta 30 dakika için karışımın inkübe edin. </li><li> 1.5 ml PBS tamponu ile iki kere yıkayın karışım. Süpernatant kaldırmak için 1 dakika boyunca 1.000 xg&#39;de santrifüjleyin. </li><li> 1 ml serum fizyolojik içine RBCs Askıya ve 4 ml akım BD flow sitometri tüpüne süspansiyonu aktarın. </li><li> FACSCanto II flo kullanılarak analizw sitometresinin, RBC kontrol kapısı üzerinde 5000 olayları edinme ve analiz için tüm olayları kullanın. </li></ol> VI. Flow sitometri ölçümleri - CD47 ekspresyonu <ol><li> 1.54 kontrol ul ya da 44 ul toplam hacim ile% 0.5 BSA ile takviye PBS tamponu ile üç kopya halinde HPG değiştirilmiş eritrositler (% 20 hematokrit) ekleyin. </li><li> BD Biosciences (New Jersey, ABD) &#39;den satın fare anti-insan CD47 etiketli phycoerythrin (PE) ve 6 ul ekle. </li><li> Bir su banyosu içinde 37 ° C sıcaklıkta 30 dakika için karışımın inkübe edin. </li><li> Iki kere 1.5 ml PBS tamponu ile karışım yıkayın. Süpernatant kaldırmak için 1 dakika boyunca 1.000 xg&#39;de santrifüjleyin. </li><li> 1 ml serum fizyolojik içine RBCs Askıya ve hücre topaklanma en aza indirmek için 26 G 5/8 iğne geçer. </li><li> 4 ml akım BD flow sitometri tüpüne süspansiyonu aktarın. </li><li> RBC kumanda ızgarası üzerinde 10.000 olayları edinme, FACSCanto II akış sitometresinin kullanarak analiz edin. </li></ol>

<ol>
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Çıkar çatışması ilan etti.

Evrensel donör eritrositler kan transfüzyonu tedavisi için artan kan kullanılabilirliği ve güvenliği büyük bir potansiyele sahiptir. Eritrosit aynı zamanda uzun sirkülasyon ve doğal biyolojik uyumluluk, 14, 15 bağlı olarak umut verici bir ilaç verme araçları olarak kabul edilir. Bu yazıda sunulan deneyler modifiye RBCs HPG in vitro özellikleri değerlendirir. In vitro özelliklerine ve HPG modifiye RBCs vivo dolaşımda son 8, 11 bizim grupta incelenmi?...

<table border="1"><tbody><tr><td> Glisidol </td><td> Sigma Aldrich </td><td></td><td> (ON, Kanada) </td></tr><tr><td> Trimetilolpropan </td><td> Fluka </td><td></td><td> (ON, Kanada) </td></tr><tr><td> Potasyum metilat </td><td> Sigma Aldrich </td><td></td><td> (ON, Kanada) </td></tr><tr><td> Susuz piridin </td><td> Sigma Aldrich </td><td></td><td> (ON, Kanada) </td></tr><tr><td> 4-Dimetilaminopiridin </td><td> Sigma Aldrich </td><td></td><td> (ON, Kanada) </td></tr><tr><td> Süksinik anhidrit </td><td> Sigma Aldrich </td><td></td><td> (ON, Kanada) </td></tr><tr><td> Aseton </td><td> Fisher Scientific </td><td></td><td> (ON, Kanada) </td></tr><tr><td> Susuz dimetil formamid </td><td> Sigma Aldrich </td><td></td><td> (ON, Kanada) </td></tr><tr><td> N-hidroksisüksinimid </td><td> Sigma Aldrich </td><td></td><td> (ON, Kanada) </td></tr><tr><td> N, N&#39;-diizopropilkarbodiimid </td><td> Sigma Aldrich </td><td></td><td> (ON, Kanada) </td></tr><tr><td> MTS kartları </td><td></td><td></td><td> Micro Typing System (MTS) kartlar (FL, ABD) </td></tr><tr><td> Dekstran 500 kDa </td><td> Pharmacia Fine Chemicals </td><td></td><td> (İsveç) </td></tr><tr><td> PEG 8 kDa </td><td> Sigma Aldrich </td><td></td><td> (ON, Kanada) </td></tr><tr><td> FITC monoklonal anti-Rh D (Rh) </td><td> Bölüm Biodiagnostics </td><td></td><td> (PA, ABD) </td></tr><tr><td> PE monoklonal anti-CD47 </td><td> BD Biosciences </td><td></td><td> (NJ, ABD) </td></tr><tr><td> Drabkin reaktifi </td><td> Sigma Aldrich </td><td></td><td> (ON, Kanada) </td></tr><tr><td></td><td></td><td></td><td> Tablo. RBC membran ve onların analizi polimerlerin HPG aşılama için kullanılan kimyasallar ve reaktifler. </td></tr></tbody></table>

antigens protected functional red blood cells by the membrane grafting of compact hyperbranched polyglycerols

Kırmızı kan hücresi (RBC) transfüzyonu gibi talasemi ve orak hücreli anemi 1-3 gibi akut ve kronik sağlık sorunları bir dizi tedavi için hayati önem taşımaktadır. RBC yüzey (~ 308 bilinen antijen 4), kronik kan transfüzyonu hastalarda eritrosit transfüzyonu, 4, 5, küçük antijen bayan maçı nedeniyle alloantibodies geliştirmek üzerinde çok sayıda antijen varlığı nedeniyle. Polietilen glikol (PEG) ve hyperbranched poligliserol (HPG) gibi hidrofilik polimerler eklenme oksijen, glukoz, ve iyon 3 gibi küçük moleküllerin geçit etkilemeden yüzey antijenleri ile antikorların etkileşimi engelleyen RBC zar üzerinde bir dışlama bir tabaka oluşturur. Şu anda herhangi bir yöntem çünkü RBC yüzey ve D antijenlerinin çok sayıda (göre protein ve karbonhidrat) varlığı tarafından sunulan zor bir meydan okuma parçası olarak evrensel verici kırmızı kan hücrelerinin üretimi için kullanılabilirBu tür yöntemlerin evelopment anlamlı transfüzyon güvenliği artırmak ve dramatik RBCs durumu ve kullanımı artıracaktır. Bu raporda, HPG ve karakterizasyonunu greftleme membran antijen korunan fonksiyonel RBCs geliştirmek için kullanılan deneyler sunulmuştur. HPGS son derece kompakt polimerler 6, 7, biyo-uyumlu, ve lipid membran 8, 9 ve maske RBC yüzey antijenleri 10, 11, çevreleyen hücre glycocalyx içinde bulunması beklenmektedir.

Rh D antijen CD47 ve RBC yüzey proteini Kamuflaj floresan işaretli monoklonal antikorlar kullanılarak akış sitometresi ile ölçüldü, ve bir temsili sonucu, Şekil 1 &#39;de verilmiştir. HPG-aşılı eritrositler durumunda (gri), sinyalin yoğunluğu yüzeyi proteinlerini maskeleme belirtmek hücre yüzeyine bağlanarak antikorlar bir azalma gösteren kontrol eritrositler (kırmızı ve yeşil) ile karşılaştırıldığında (sola doğru kayar tepe noktası) azalmıştır . <p class="jove_...

Watch this Scientific Journal Video about Antigens Protected Functional Red Blood Cells By The Membrane Grafting Of Compact Hyperbranched Polyglycerols at JoVE.com

Antigens Protected Functional Red Blood Cells By The Membrane Grafting Of Compact Hyperbranched Polyglycerols

Hyperbranched poligliserol (HPG) ile kırmızı kan hücrelerinin hücre zarı modifikasyonu (eritrositler) sunulmuştur. Modifiye RBCs sulu iki faz bölümleme, osmotik frajilitesi ve kompleman aracılı lizis ile karakterize edildi. Yüzey proteinleri ve antijenlerin kamuflaj akım sitometri ve Mikro Yazma Sistemi (MTS) kan fenotipleme kartları kullanılarak değerlendirilmiştir.

Antijenler Kompakt Hyperbranched Polyglycerols Of Aşılama Membran By Fonksiyonel Kırmızı Kan Hücreleri Korumalı

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell ...

antigens-protected-functional-red-blood-cells-membrane-grafting

Research

JoVE Journal

Immunology and Infection

18.0K Görüntüleme.  University of British Columbia. Hyperbranched poligliserol (HPG) ile kırmızı kan hücrelerinin hücre zarı modifikasyonu (eritrositler) sunulmuştur. Modifiye RBCs sulu iki faz bölümleme, osmotik frajilitesi ve kompleman aracılı lizis ile karakterize edildi. Yüzey proteinleri ve antijenlerin kamuflaj akım sitometri ve Mikro Yazma Sistemi (MTS) kan fenotipleme kartları kullanılarak değerlendirilmiştir.

Video: Antigens Protected Functional Red Blood Cells By The Membrane Grafting Of Compact Hyperbranched Polyglycerols

Depletion of Specific Cell Populations by Complement Depletion

The purification of immune cell populations is often required in order to study their unique functions. In particular, molecular approaches such as real-time PCR and microarray analysis require the isolation of cell populations with high purity. Commonly used purification strategies include fluorescent activated cell sorting (FACS), magnetic bead separation and complement depletion. Of the three strategies, complement depletion offers the advantages of being fast, inexpensive, gentle on the cells and a high cell yield. The complement system is composed of a large number of plasma proteins that when activated initiate a proteolytic cascade culminating in the formation of a membrane-attack complex that forms a pore on a cell surface resulting in cell death1. The classical pathway is activated by IgM and IgG antibodies and was first described as a mechanism for killing bacteria. With the generation of monoclonal antibodies (mAb), the complement cascade can be used to lyse any cell population in an antigen-specific manner. Depletion of cells by the complement cascade is achieved by the addition of complement fixing antigen-specific antibodies and rabbit complement to the starting cell population. The cells are incubated for one hour at 37&deg;C and the lysed cells are subsequently removed by two rounds of washing. MAb with a high efficiency for complement fixation typically deplete 95-100% of the targeted cell population. Depending on the purification strategy for the targeted cell population, complement depletion can be used for cell purification or for the enrichment of cell populations that then can be further purified by a subsequent method.

To effectively study the function of immune cell populations their purification is often required. Complement depletion is a fast and inexpensive technique for the isolation of immune cell populations with high purity.

Kompleman tükenmesi Özel Hücre Populasyonlarının tükenmesi

The purification of immune cell populations is often required in order to study their unique functions.  In particular, molecular approaches such as ...

İmmünoloji ve Enfeksiyon

Isolation of Mouse Peritoneal Cavity Cells

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal tract and other viscera. It harbors a number of immune cells including macrophages, B cells and T cells. The presence of a high number of na&iuml;ve macrophages in the peritoneal cavity makes it a preferred site for the collection of na&iuml;ve tissue resident macrophages (1). The peritoneal cavity is also important to the study of B cells because of the presence of a unique peritoneal cavity-resident B cell subset known as B1 cells in addition to conventional B2 cells. B1 cells are subdivided into B1a and B1b cells, which can be distinguished by the surface expression of CD11b and CD5. B1 cells are an important source of natural IgM providing early protection from a variety of pathogens (2-4). These cells are autoreactive in nature (5), but how they are controlled to prevent autoimmunity is still not understood completely. On the contrary, CD5+ B1a cells possess some regulatory properties by virtue of their IL-10 producing capacity (6). Therefore, peritoneal cavity B1 cells are an interesting cell population to study because of their diverse function and many unaddressed questions associated with their development and regulation. The isolation of peritoneal cavity resident immune cells is tricky because of the lack of a defined structure inside the peritoneal cavity. Our protocol will describe a procedure for obtaining viable immune cells from the peritoneal cavity of mice, which then can be used for phenotypic analysis by flow cytometry and for different biochemical and immunological assays.

The peritoneal cavity in mammals contains different immune cell populations crucial for innate immune responses. An efficient isolation method is required for biochemical and functional analyses of these cells. Here we provide a comprehensive method for the isolation of peritoneal cavity cells in the mouse.

Fare Periton Boşluk Hücreleri İzolasyon

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal ...

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS)

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known phenotype.  Other bulk methods of purification include panning, complement depletion and magnetic bead separation.  However, FACS has several advantages over other available methods.  FACS is the preferred method when very high purity of the desired population is required, when the target cell population expresses a very low level of the identifying marker or when cell populations require separation based on differential marker density.  In addition, FACS is the only available purification technique to isolate cells based on internal staining or intracellular protein expression, such as a genetically modified fluorescent protein marker.  FACS allows the purification of individual cells based on size, granularity and fluorescence.  In order to purify cells of interest, they are first stained with fluorescently-tagged monoclonal antibodies (mAb), which recognize specific surface markers on the desired cell population (1).  Negative selection of unstained cells is also possible.  FACS purification requires a flow cytometer with sorting capacity and the appropriate software.  For FACS, cells in suspension are passed as a stream in droplets with each containing a single cell in front of a laser.  The fluorescence detection system detects cells of interest based on predetermined fluorescent parameters of the cells.  The instrument applies a charge to the droplet containing a cell of interest and an electrostatic deflection system facilitates collection of the charged droplets into appropriate collection tubes (2).  The success of staining and thereby sorting depends largely on the selection of the identifying markers and the choice of mAb.  Sorting parameters can be adjusted depending on the requirement of purity and yield.  Although FACS requires specialized equipment and personnel training, it is the method of choice for isolation of highly purified cell populations.

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting FACS

For many scientific studies requiring a biological and chemical analysis of cell populations the cells must be in a high state of purity. Fluorescence activated cell sorting (FACS) is a superior method in which to obtain pure cell populations.

Floresan Aktive Hücre Ayırma (FACS) Özgül Hücre Nüfus Arıtma

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known ...

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of NK cells and ability to expand in vitro has restricted development of NK cell immunotherapy. Here we describe a method to efficiently expand vast quantities of functional NK cells ex vivo using K562 cells expressing membrane-bound IL21, as an artificial antigen-presenting cell (aAPC).
NK cell adoptive therapies to date have utilized a cell product obtained by steady-state leukapheresis of the donor followed by depletion of T cells or positive selection of NK cells. The product is usually activated in IL-2 overnight and then administered the following day 1. Because of the low frequency of NK cells in peripheral blood, relatively small numbers of NK cells have been delivered in clinical trials.
The inability to propagate NK cells in vitro has been the limiting factor for generating sufficient cell numbers for optimal clinical outcome. Some expansion of NK cells (5-10 fold over 1-2 weeks) has be achieved through high-dose IL-2 alone 2. Activation of autologous T cells can mediate NK cell expansion, presumably also through release of local cytokine 3. Support with mesenchymal stroma or artificial antigen presenting cells (aAPCs) can support the expansion of NK cells from both peripheral blood and cord blood 4. Combined NKp46 and CD2 activation by antibody-coated beads is currently marketed for NK cell expansion (Miltenyi Biotec, Auburn CA), resulting in approximately 100-fold expansion in 21 days.
Clinical trials using aAPC-expanded or -activated NK cells are underway, one using leukemic cell line CTV-1 to prime and activate NK cells5 without significant expansion. A second trial utilizes EBV-LCL for NK cell expansion, achieving a mean 490-fold expansion in 21 days6. The third utilizes a K562-based aAPC transduced with 4-1BBL (CD137L) and membrane-bound IL-15 (mIL-15)7, which achieved a mean NK expansion 277-fold in 21 days. Although, the NK cells expanded using K562-41BBL-mIL15 aAPC are highly cytotoxic in vitro and in vivo compared to unexpanded NK cells, and participate in ADCC, their proliferation is limited by senescence attributed to telomere shortening8. More recently a 350-fold expansion of NK cells was reported using K562 expressing MICA, 4-1BBL and IL159.
Our method of NK cell expansion described herein produces rapid proliferation of NK cells without senescence achieving a median 21,000-fold expansion in 21 days.

Here we describe a method to efficiently expand and purify large numbers of human NK cells and assess their function.

Genişleme, Saflaştırılması, ve İnsan Periferik Kan NK Hücrelerinin Fonksiyonel Değerlendirme

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

Methods for Quantitative Detection of Antibody-induced Complement Activation on Red Blood Cells

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver (extravascular hemolysis) or leading to intravascular lysis of RBCs. Alloantibodies (e.g. ABO) or autoantibodies to RBC antigens (as seen in autoimmune hemolytic anemia, AIHA) leading to complement activation are potentially harmful and can be - especially when leading to intravascular lysis - fatal1. Currently, complement activation due to (auto)-antibodies on RBCs is assessed in vitro by using the Coombs test reflecting complement deposition on RBC or by a nonquantitative hemolytic assay reflecting RBC lysis1-4. However, to assess the efficacy of complement inhibitors, it is mandatory to have quantitative techniques. Here we describe two such techniques. First, an assay to detect C3 and C4 deposition on red blood cells that is induced by antibodies in patient serum is presented. For this, FACS analysis is used with fluorescently labeled anti-C3 or anti-C4 antibodies. Next, a quantitative hemolytic assay is described. In this assay, complement-mediated hemolysis induced by patient serum is measured making use of spectrophotometric&#160;detection of the released hemoglobin. Both of these assays are very reproducible and quantitative, facilitating studies of antibody-induced complement activation.

Here we describe two assays for measuring complement activation induced by antibodies against red blood cells. The major advantage over the current assays is their quantitative and easy-to-interpret nature.

Kırmızı kan hücreleri üzerinde antikor kaynaklı kompleman aktivasyon sayısal tespiti için Yöntemler

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver ...

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. ApcMin/+ mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment.

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model.

Miyeloid hücrelerin Dynamics Gerçek zamanlı Görüntüleme<em&gt; Süm<sup&gt; Min / +</sup</em&gt; Bağırsak Tümörleri Disk Eşodaklı Mikroskopi Spinning tarafından

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques ...

Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. Polysaccharides such as glycogen, glycoproteins, and glycolipids stain bright magenta making it easy to enumerate positive and negative cells within the tissue. In muscle cells PAS staining is used to determine the glycogen content in different types of muscle cells, while in blood cell samples PAS staining has been explored as a diagnostic tool for a variety of conditions. Blood contains a proportion of white blood cells that belong to the immune system. The notion that cells of the immune system possess glycogen and use it as an energy source has not been widely explored. Here, we describe an adapted version of the PAS staining protocol that can be applied on peripheral blood mononuclear immune cells from human venous blood. Small cells with PAS-positive granules and larger cells with diffuse PAS staining were observed. Treatment of samples with amylase abrogates these patterns confirming the specificity of the stain. An alternate technique based on enzymatic digestion confirmed the presence and amount of glycogen in the samples. This protocol is useful for hematologists or immunologists studying polysaccharide content in blood-derived lymphocytes.

Periodic acid Schiff staining is a technique that visualizes the polysaccharide content of tissues. This article demonstrates periodic acid Schiff staining protocol adapted for use on peripheral blood mononuclear cells purified from human venous blood. Such samples are enriched for lymphocytes and other white blood cells of the immune system.

Periyodik Asit Schiff Boyama ile periferik kan mononükleer hücreleri glikojen algılama

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. ...

Development of an Antigen-driven Colitis Model to Study Presentation of Antigens by Antigen Presenting Cells to T Cells

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological mechanisms involved during the disease is the T cell transfer model of colitis. In this model, immunodeficient mice (RAG-/- recipients) are reconstituted with naive CD4+ T cells from healthy wild type hosts.
This model allows examination of the earliest immunological events leading to disease and chronic inflammation, when the gut inflammation perpetuates but does not depend on a defined antigen. To study the potential role of antigen presenting cells (APCs) in the disease process, it is helpful to have an antigen-driven disease model, in which a defined commensal-derived antigen leads to colitis. An antigen driven-colitis model has hence been developed. In this model OT-II CD4+ T cells, that can recognize only specific epitopes in the OVA protein, are transferred into RAG-/- hosts challenged with CFP-OVA-expressing E. coli. This model allows the examination of interactions between APCs and T cells in the lamina propria.

In this antigen-driven colitis model, OT-II CD4+ T cells expressing a red fluorescent protein were adoptively transferred into RAG-/- mice that express a green fluorescent protein in mononuclear phagocytes (MPs). The hosts were challenged with Escherichia coli (E.coli) expressing the ovalbumin protein (OVA) fused to a cyan fluorescent protein (CFP).

Bir Antijen odaklı Kolit Modelinin Geliştirilmesi Antijen T hücreleri için Hücreler sunulması ile Antijen Sunumu Eğitim için

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological ...

Using X-ray Crystallography, Biophysics, and Functional Assays to Determine the Mechanisms Governing T-cell Receptor Recognition of Cancer Antigens

Human CD8+ cytotoxic T lymphocytes (CTLs) are known to play an important role in tumor control. In order to carry out this function, the cell surface-expressed T-cell receptor (TCR) must functionally recognize human leukocyte antigen (HLA)-restricted tumor-derived peptides (pHLA). However, we and others have shown that most TCRs bind sub-optimally to tumor antigens. Uncovering the molecular mechanisms that define this poor recognition could aid in the development of new targeted therapies that circumnavigate these shortcomings. Indeed, present therapies that lack this molecular understanding have not been universally effective. Here, we describe methods that we commonly employ in the laboratory to determine how the nature of the interaction between TCRs and pHLA governs T-cell functionality. These methods include the generation of soluble TCRs and pHLA and the use of these reagents for X-ray crystallography, biophysical analysis, and antigen-specific T-cell staining with pHLA multimers. Using these approaches and guided by structural analysis, it is possible to modify the interaction between TCRs and pHLA and to then test how these modifications impact T-cell antigen recognition. These findings have already helped to clarify the mechanism of T-cell recognition of a number of cancer antigens and could direct the development of altered peptides and modified TCRs for new cancer therapies.

Here, we describe methods that we commonly employ in the laboratory to determine how the nature of the interaction between the T-cell receptor and tumor antigens, presented by human leukocyte antigens, governs T-cell functionality; these methods include protein production, X-ray crystallography, biophysics, and functional T-cell experiments.

X-ışını kristalografisi, biyofizik ve fonksiyonel tahliller kullanılarak mekanizmalar Yönetim T hücresi kanser antijenleri Reseptör tanıma belirlemek için

Human CD8+ cytotoxic T lymphocytes (CTLs) are known to play an important role in tumor control. In order to carry out this function, the cell ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

Dextran fare ve insan birincil NK hücreleri Lentiviral iletim verimliliğini artırır

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...