Yazarlar Savunma Bakanlığı aracılığıyla Congressionally Yönetmen Tıbbi Araştırma Programları (# W81XWH-10-1-0445), Ulusal Sağlık Enstitüleri (NIH R21 HL110056) ve Amerikan Kalp Derneği (# 11SDG4890030) finanse edersiniz.

1. Tamponlar ve Test Aracıları Hazırlanması ve Sterilizasyonu <ol><li> 150 mM NaCl tampon: nanopure 500 ml su içinde 4.383 g NaCl kristaller eritilir. </li><li> pH Tamponları: monobazik ve dibazik sodyum fosfat uygun miktarlarda karıştırılarak pH 5.6, 6.2, 6.8 ve 7.4 &#39;de fosfat tamponlar hazırlayın. Örnekleri gibi sitrat tampon olarak daha sonra düşük pH değerleri (yani pH &lt;5.6) daha uygun bir tampon, test edilecek ise, kullanılmalıdır. Tampon tarifleri kolayca kullanılabilir ve bir örnek burada referans verilmiştir. 12 </li><li> Bir şişe üstü vakumlu filtrasyon aparatı ve yeniden kontrol tampon pH aracılığıyla yukarıda belirtilen tüm tamponları sterilize edin. </li><li> % 20 oranında Triton X-100 (pozitif kontrol): Karışım 20 ml saf Triton nanopure 80 ml su içinde X-100. Şiddetle Vortex ve çözülmekte sonikasyon. Kullanımdan önce gece boyunca oda sıcaklığında bırakın. </li></ol> 2. Eritrositler hazırlanması <ol><li> Kan fr 25 ml eldepıhtılaşmayı önlemek için K 2-EDTA kaplı Vacutainer tüpler içine doğrudan çizilmiş om anonim bir insan donör,. </li></ol> NOT: Tüm işlemler uygun Kurumsal İnceleme Kurulu (KİK), ve Damara ve kan toplama tarafından önceden onaylanmalıdır donör riskini en aza indirmek için eğitimli bir Phlebotomist tarafından yapılmalıdır. Standart flebotomi prosedürler yerde yayımlanmamış edilmiştir. 13. <ol start="2"><li> 5 dk, ve hematokrit düzeyleri işareti (kırmızı, alt tabaka) ve plazma (sarımsı, üst tabaka) tüp için 500 xg&#39;de kan santrifüjleyin. </li><li> Bir mikropipet ile hafifçe plazma aspire, çamaşır suyu içine ekleyin ve biyolojik tehlikeli atık içine atın. </li><li> 150 mM NaCl çözeltisi ile işaretlenmiş hattı (plazma orijinal seviyesi) hematokrit tüpü doldurun. Cap ve hafifçe karıştırmak için birkaç kez ters. 5 dakika boyunca 500 xg&#39;de santrifüjleyin. </li><li> Tekrar kan hücreleri yıkayın adım 2,3-2,4 tekrarlayın. Sonra supernat aspirekarınca ve pH 7.4 &#39;de PBS ile değiştirin. Karıştırmak için Invert. </li><li> Test edilecek her bir pH tekabül, eşit dört tüpler içine kan Böl. Test edilecek her bir pH (5.6, 6.2, 6.8, 7.4) &#39;ye göre tüp etiketleyin. </li><li> 5 dakika boyunca 500 xg&#39;de kan tüpleri santrifüjleyin. Borular üzerinde seviyeleri işaretleyin, sonra süpernatant aspire. </li><li> Uygun pH (as 2.6 belirtilir) tamponu ile işaretlenmiş satırı her tüp doldurun. </li><li> Etiket dört 50 ml konik tüpler (test pH başına bir), ve her konik tüp içine uygun pH PBS pipet 49 ml. </li><li> Bir 1:50 seyreltme için gelen tüpe eritrositler (aynı pH) 1 ml ekleyin. Görme bulanık olmalıdır ve bozulmamış bırakılırsa razı olacak seyreltilmiş kan, kontrol edin. Hiçbir pelet formlarda, hücreler lize var. </li></ol> 3. Lizis Testi 96 Şey Levha Kurulumu ve Sayısallaştırma <ol><li> Test edilecek 20x arzu edilen nihai konsantrasyonu, bütün deneysel ilaç aktarma maddelerinin stok çözelti hazırlayın (Assayilaç dağıtım ajan 10 ul + nihai test karışımının içine orijinal ilaç dağıtım aracı bir 1/20 dilüsyon giden 190 ul seyreltilmiş kırmızı kan hücreleri,) alacaktır. 20, 100, ve 800 ug / ml &#39;lik Stoklar, sırasıyla 1, 5, ve 40 ug / mL, nihai deney konsantrasyonu ile sonuçlanır önerilmektedir. </li><li> V-dibine her stok solüsyonu 96-kuyucuğu pipetleyin 10 ul. En iyi sonuçlar için, üç kopya halinde her bir numune yüklemek veya dört misli. </li></ol> NOT: kolaylığı için, ayrı bir 96-plaka plaka başına n&#39;deki = 3-4 yüklenen (her konsantrasyonda) Her numune ile test edilecek her pH için hazırlıklı olmak gerektiğini tavsiye edilir. <ol start="3"><li> Pozitif kontrol kuyuları,% 20 Triton X-100, 10 ul ilave edin. </li><li> Negatif kontrol kuyuları fosfat tamponu 10 ul ilave edin. Test edilecek aynı pH tampon kullanın. </li><li> Seyreltilmiş Eritrositlerin Pipet 190 ul her oyuğa (2.10 &#39;a bakın), kalır emin hücre stok solüsyonu yapmatransfer sırasında homojen. İpucu: Bu görevi kolaylaştırmak için kullanın çok kanallı pipet. </li><li> Bir saat (: orbital çalkalayıcı ya da rocker kullanın Opsiyonel) için 37 ° C&#39;de plakaları inkübe edin. </li><li> 500 pelet sağlam eritrositlere xg de 5 dakika plakaları santrifüjleyin. Not: santrifüj plaka kaldırılarak ve bir sonraki adım için taşırken, dikkatli olun ve hücre pelletini bozmaya kesin olmayacak. </li><li> Bir çok kanallı pipet kullanarak, açık, düz tabanlı 96-plaka içine her kuyudan süpernatant 100 ul aktarın. Not: hücre pelletini yanlışlıkla herhangi bir numune (ler) için rahatsız ise, bir süpernatant transferi işlemlerine ardından plakayı tekrar santrifüj ve. </li><li> Bir plaka okuyucu ile yüzer maddelerinin absorbansı ölçülür. Bir dalga boyu aralığı (- 541 nm ilâ 400) kullanılabilir dikkat ediniz. </li></ol> NOT: Farklı plaka okuyucu farklı doygunluk noktaları ve hassasiyetleri olabilir, bu yüzden benim için bir dalga boyu seçimiasurements deterjan tedavisinin yol açtığı gibi deneysel örneklerinden hemoliz veriler güvenilir maksimum hemoliz karşı normalize edilebilir olup olmadığına bağlıdır. Bu pozitif kontrol numunelerinin absorbans değerlerinin doğru bir ölçüm gerektirir. <ol start="10"><li> Microsoft Excel veya benzer bir veri analiz yazılımı kullanarak, her bir pH (adım 3.4) için ayarlanmış negatif kontrol örnekleri arka absorbans ölçümleri ortalama bulabilirsiniz. Bu pH ölçüldü diğer tüm numuneler bu plan absorbans Değ.Çıkar. </li><li> Arka plan çıkarma sonra, pozitif kontrol ortalama absorbans deterjan işlenmiş örnekleri (adım 3.3) bulabilirsiniz. Sonra% 100 hemoliz temsil etmelidir ki, bu ortalama absorbans değeri tüm deneysel veri noktaları normalleştirmek. Nihayet, deterjan kontrolü iyi göreli her oluştu% hemoliz hesaplamak için% 100 her iyi değeriyle çarpın. </li></ol>

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IRB onayı: İnsan denekleri Usul Vanderbilt Üniversitesi Kurumsal İnceleme Kurulu (KİK; Protokolü # 111251) tarafından onaylanmıştır. Çıkar çatışması ilan etti.

endosomolitik fonksiyon için tasarlanmış pH duyarlı polimerler ya da diğer maddeler gibi hızla ve etkili bir şekilde endosome karşılaşılan pH değerlerinde kırmızı kan hücrelerinin lizisi (Şekil 1 esas taranmış olabilir; pH 6.8 - erken endosome, pH 6.2 - geç endosome, pH 5,6 - Lizozom). 14-17 pH bağımlı hemoliz biomacromolecular tedavi (örn. peptidler, siRNA, ODN&#39;ler, proteinler) endozomal serbest arabuluculuk taşıyıcılar yeteneği taranması için kull...

Hücre içinde birçok potansiyel yüksek etkili tedavi hedefleri olmasına rağmen, maddelerin hücre içi teslimat önemli bir sorun teşkil etmektedir. Sık sık, ilaçlar, özellikle biyolojik, ekzositoz yoluyla hücreler tarafından içselleştirilmesi ve veziküller içine kaçırılan ya endo-lizozom yolağı yoluyla içindekiler bozulmaya yol açan veya hücrenin geri shuttled vardır vardır. Ikincisi süreçte 1, iç pH veziküllerin enzim aktivitesi için optimum pH olan yaklaşık olarak 5-6 olana kadar asitleştirildi, bu gibi lisozim gibi bu bölme içinde işlev. 2 Son zamanlarda, malzemeleri bir dizi özel bir asitleştirme kendi kargo sitozolik teslim kolaylaştırmak için endozomlar kaldıraç için tasarlanmış edilmiştir. Bu yaklaşımın bir örneği olan temel fizyolojik pH (yani 7.4) zwitteriyonik ve şarj nötr sentetik polimer misel nanopartiküller kullanır. Bununla birlikte, pH 6.0 &#39;- 6.5, polimerler protonlanır olmak ve misel çekirdek oynattığını net pozitif yük kazanma, ve maruz kalan polimer segmentleri ile etkileşim ve endozomal zarı bozar. Bu aktivite, onların sitosolik hedefleri erişim imkanı sağlayan, peptid ve nükleik asit-bazlı terapötiklerin endozomal kaçış teşvik etmek için gösterilmiştir. Membran bariyeri bozmak endozomal kaçış aracılık için geliştirilmiş yöntemlerin diğer örnekleri ya da 3,4 &#39;füzojenik&#39; peptitler yer fosfolipid çift-katlı membran füzyonu veya geçici gözenek oluşumuna aracılık eden proteinlerdir. örneğin poli (propylacrylic asit) gibi anyonik alkil akrilik asit homopolimerleri 5 diğer bir iyi incelenmiş olan bir yaklaşım, ve bu polimerler de, kolye karboksilik asit protonasyon durum geçiş belirler Endo-Lizozomal pH aralıkları bir hidrofobik, membran-yıkıcı devlet. 6,7 Endosomolitik davranışı tarama için yararlı bir model sistem ex in vivo pH bağımlı hemoliz testi. 8 Bu model sistem olarak, eritrosit membranı endo-lizozomal veziküller içine iki tabakalı lipid membran için bir vekil olarak hizmet vermektedir. Bu genellenebilir modeli hücre delici peptidler ve diğer polimerik gen aktarım sistemlerinin endosomolitik davranışlarını değerlendirmek için başkaları tarafından kullanılmaktadır. 8-11 Bu deneyde, insan kırmızı kan hücreleri ve test materyalleri taklit tanımlanan pH&#39;da tampon birlikte kuluçkaya vardır ekstraselüler (7.4), erken (6.8) endozomal ve endo-lizozomal geç (&lt;6.8) ortamları. Kuluçka dönemi sırasında serbest hemoglobin miktar deterjan ile lize pozitif kontrol örneklerinde serbest hemoglobin miktar ile normalize edilir kırmızı kan hücrelerinin lizisi, bir ölçüsü olarak ölçülür. Potansiyel endosomolitik malzemeleri test küçük bir kütüphane tarama itibaren, bir pH 7.4 hiç hemoliz üretmek örnekleri anlaması, ancak önemli ölçüde yükselmiş etek olabilirpH &lt;6.5 olan olysis, sitozolik ilaç dağıtım için en etkili ve cytocompatible adayları olacak. Bu kriterlere uyan malzemeler gelişigüzel endo-lizozomal bölmeye içselleştirilmesi sonrasında yerel pH bir düşüş maruz kadar iki tabakalı lipid membranlar (sitotoksisite neden olabilir yani) yok eylemsiz değil kalması beklenir. Bu protokolde, eritrositler bir insan donör izole edilir ve pH 5.6, 6.2, 6.8, ya da deneysel endosomolitik ilaç dağıtım ajanları ile 7.4 &#39;de co-inkübe. Bozulmamış eritrositler peletlenmiş, ve süpernatan (erimiş eritrositler serbest hemoglobin ihtiva eden) bir plaka okuyucu (Şekil 1) vasıtasıyla hemoglobin karakteristik absorbans için analiz edilir.

<table border="1"><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar (isteğe bağlı) </td></tr><tr><td> BD Vacutainer - K2EDTA Vacutainer Tüpler </td><td> Fisher Scientific </td><td> 22-253-145 </td><td> Kan toplama için </td></tr><tr><td> BD Vacutainer Kan Toplama İğneler, 20.5-ölçer </td><td> Fisher Scientific </td><td> 02-665-31 </td><td> Kan toplama için </td></tr><tr><td> BD Vacutainer Tüp Tutucu / İğne Adaptörü </td><td> Fisher Scientific </td><td> 22-289-953 </td><td> Kan toplama için </td></tr><tr><td> BD Marka İzopropil Alkol Swablar </td><td> Fisher Scientific </td><td> 13-680-63 </td><td> Kan toplama için </td></tr><tr><td> BD Vacutainer Lateks-Alerjik Turnike </td><td> Fisher Scientific </td><td> 02-657-6 </td><td> Kan toplama için </td&gt; </tr><tr><td> Hidroklorik asit (kons.) </td><td> Fisher Scientific </td><td> A144-500 </td><td> D-PBS, pH ayarlaması için. </td></tr><tr><td> Triton X-100 </td><td> Sigma-Aldrich </td><td> T8787 </td><td> Pozitif kontrol </td></tr><tr><td> Dulbecco PBS </td><td> Invitrogen </td><td> 14190 </td><td></td></tr><tr><td> SFCA Membranlı Nalgene MF75 Steril Disposable Şişe Üstü Filtre Ünitesi </td><td> Fisher Scientific </td><td> 09-740-44A </td><td></td></tr><tr><td> BD 96-kuyucuğu, düz dipli, doku kültürü ile tedavi edilen polistiren </td><td> Fisher Scientific </td><td> 08-772-2C </td><td> Testin sonunda, plaka okuma için. </td></tr><tr><td> BD 96-kuyucuğu, yuvarlak dipli, doku kültürü ile tedavi edilen polistiren </td><td> Fisher Scientific </td><td> 08-772-17 </td><td> Deneysel ajanlar ile kırmızı kan hücrelerinin kuluçka için. </td></tr></table&gt;

ex vivo red blood cell hemolysis assay for the evaluation of ph-responsive endosomolytic agents for cytosolic delivery of biomacromolecular drugs

Endo-lizozomal veziküller oluşturan çift katmanlı fosfolipidlerin hücre içi hedeflere biyolojik ilaçların teslim için bir engel teşkil edebilir. Bu engeli aşmak için, sentetik ilaç taşıyıcı bir dizi aktif endozomal zarı bozar ve sitoplazma içine kargo sunmak için tasarlanmış edilmiştir. Burada, hücre içi ilaç dağıtım sistemleri cytocompatibility ve endosomolitik etkinlik için hızlı, yüksek hacimli ekran olarak kullanılabilir hemoliz tahlil, açıklar. Hemoliz testi, insan kırmızı kan hücreleri ve test materyalleri ekstraselüler, erken endozomal ve geç endo-lizozomal ortamlarda taklit tanımlanan pH&#39;da tampon co-inkübe edilir. Pelet bozulmamış kırmızı kan hücreleri santrifüj basamağı sonrasında, orta salınan hemoglobin miktarı, spektrofotometrik (en iyi dinamik aralığı için 405 nm) olarak ölçülür. Yüzde kırmızı kan hücre parçalama sonra pozitif kontrol samp göre ölçülürles bir deterjan ile lize. Bu model sistemde eritrosit membran endo-lizozomal veziküller içine iki tabakalı lipid membran için bir vekil olarak hizmet vermektedir. İstenen sonucu fizyolojik pH (7.4) ve yaklaşık pH 5-6,8 gelen endo-lizozom pH aralığında sağlam hemoliz de ihmal hemoliz olduğunu.

<html> Genellikle, ideal bir pH bağımlı hemolitik davranışı gösteren ilaç ajanları, nükleik asitler ya da biyolojik olarak aktif diğer moleküllerin sitosolik teslimat için yüksek bir potansiyele sahiptir. Bu pH 7.4 &#39;de minimal hemoliz sergiler Şekil 2&#39;de de gösterildiği gibi, Ajan # 1 örneklediği, ama endozomal pH aralıkları (&lt;6.5) de hemolitik davranış keskin bir artış olduğunu. Bu ajanlar hemocompatible olmayabilir ve potansiyel olarak bu konsantrasyonlarda sitotoks...

Watch this Scientific Journal Video about Ex Vivo Red Blood Cell Hemolysis Assay for the Evaluation of pH-responsive Endosomolytic Agents for Cytosolic Delivery of Biomacromolecular Drugs at JoVE.com

Ex Vivo Red Blood Cell Hemolysis Assay for the Evaluation of pH-responsive Endosomolytic Agents for Cytosolic Delivery of Biomacromolecular Drugs

Bir hemoliz tahlil hücre içi kargo için ilaç dağıtım sistemleri &#39;cytocompatibility ve endosomolitik aktivite hızlı, yüksek hacimli ekran olarak kullanılabilir. Tahlil çevresel pH bir fonksiyonu olarak eritrosit membran parçalama ölçer.

Biomacromolecular Uyuşturucu Sitozolik Teslimat için pH-duyarlı endosomolitik Ajanlar değerlendirilmesi için Ex Vivo Kırmızı Kan Hücresi Hemoliz Testi

Phospholipid bilayers that constitute endo-lysosomal vesicles can pose a barrier to delivery of biologic drugs to intracellular targets. To overcome this ...

ex-vivo-red-blood-cell-hemolysis-assay-for-evaluation-ph-responsive

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Immunology and Infection

Ex Vivo Red Blood Cell Hemolysis Assay for the Evaluation of pH-responsive Endosomolytic Agents for Cytosolic Delivery of Biomacromolecular Drugs

34.0K Görüntüleme.  Vanderbilt University. Bir hemoliz tahlil hücre içi kargo için ilaç dağıtım sistemleri 'cytocompatibility ve endosomolitik aktivite hızlı, yüksek hacimli ekran olarak kullanılabilir. Tahlil çevresel pH bir fonksiyonu olarak eritrosit membran parçalama ölçer.

Video: Ex Vivo Red Blood Cell Hemolysis Assay for the Evaluation of pH-responsive Endosomolytic Agents for Cytosolic Delivery of Biomacromolecular Drugs

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

Bakteriyel Yapışma Çalışma Kesme Stres Tanıtımı

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

İmmünoloji ve Enfeksiyon

A Quantitative Evaluation of Cell Migration by the Phagokinetic Track Motility Assay

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular organisms towards a source of nutrients or away from unsuitable conditions, as well as in multicellular organisms for tissue development, immune surveillance and wound healing, just to mention a few roles1,2,3. Deregulation of this process can lead to serious neurological, cardiovascular and immunological diseases, as well as exacerbated tumor formation and spread4,5. Molecularly, actin polymerization and receptor recycling have been shown to play important roles in creating cellular extensions (lamellipodia), that drive the forward movement of the cell6,7,8. However, many biological questions about cell migration remain unanswered.
The central role for cellular motility in human health and disease underlines the importance of understanding the specific mechanisms involved in this process and makes accurate methods for evaluating cell motility particularly important. Microscopes are usually used to visualize the movement of cells. However, cells move rather slowly, making the quantitative measurement of cell migration a resource-consuming process requiring expensive cameras and software to create quantitative time-lapsed movies of motile cells. Therefore, the ability to perform a quantitative measurement of cell migration that is cost-effective, non-laborious, and that utilizes common laboratory equipment is a great need for many researchers.
The phagokinetic track motility assay utilizes the ability of a moving cell to clear gold particles from its path to create a measurable track on a colloidal gold-coated glass coverslip9,10. With the use of freely available software, multiple tracks can be evaluated for each treatment to accomplish statistical requirements. The assay can be utilized to assess motility of many cell types, such as cancer cells11,12, fibroblasts9, neutrophils13, skeletal muscle cells14, keratinocytes15, trophoblasts16, endothelial cells17, and monocytes10,18-22. The protocol involves the creation of slides coated with gold nanoparticles (Au&deg;) that are generated by a reduction of chloroauric acid (Au3+) by sodium citrate. This method was developed by Turkevich et al. in 195123 and then improved in the 1970s by Frens et al.24,25. As a result of this chemical reduction step, gold particles (10-20 nm in diameter) precipitate from the reaction mixture and can be applied to glass coverslips, which are then ready for use in cellular migration analyses9,26,27.
In general, the phagokinetic track motility assay is a quick, quantitative and easy measure of cellular motility. In addition, it can be utilized as a simple high-throughput assay, for use with cell types that are not amenable to time-lapsed imaging, as well as other uses depending on the needs of the researcher. Together, the ability to quantitatively measure cellular motility of multiple cell types without the need for expensive microscopes and software, along with the use of common laboratory equipment and chemicals, make the phagokinetic track motility assay a solid choice for scientists with an interest in understanding cellular motility.

The phagokinetic motility track assay is a method used to assess the movement of cells. Specifically, the assay measures chemokinesis (random cell motility) over time in a quantitative manner. The assay takes advantage of the ability of cells to create a measurable track of their movement on colloidal gold-coated coverslips.

Phagokinetic Parça Motilite Testi sayesinde Hücre Göç Kantitatif Değerlendirilmesi

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular ...

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

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 anemia 1-3. Due to the presence of multitude of antigens on the RBC surface (~308 known antigens 4), patients in the chronic blood transfusion therapy develop alloantibodies due to the miss match of minor antigens on transfused RBCs 4, 5. Grafting of hydrophilic polymers such as polyethylene glycol (PEG) and hyperbranched polyglycerol (HPG) forms an exclusion layer on RBC membrane that prevents the interaction of antibodies with surface antigens without affecting the passage of small molecules such as oxygen ,glucose, and ions3. At present no method is available for the generation of universal red blood donor cells in part because of the daunting challenge presented by the presence of large number of antigens (protein and carbohydrate based) on the RBC surface and the development of such methods will significantly improve transfusion safety, and dramatically improve the availability and use of RBCs. In this report, the experiments that are used to develop antigen protected functional RBCs by the membrane grafting of HPG and their characterization are presented. HPGs are highly biocompatible compact polymers 6, 7, and are expected to be located within the cell glycocalyx that surrounds the lipid membrane 8, 9 and mask RBC surface antigens10, 11.

The cell membrane modification of red blood cells (RBCs) with hyperbranched polyglycerol (HPG) is presented. Modified RBCs were characterized by aqueous two phase partitioning, osmotic fragility and complement mediated lysis. The camouflage of surface proteins and antigens was evaluated using the flow cytometry and Micro Typing System (MTS) blood phenotyping cards.

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 ...

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 ...

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific locked nucleic acid (LNA) and molecular beacon (MB) probes, transcript standards, automated multichannel pipetting, and plate drying. Determining expression among the type I interferons (IFN), especially the twelve IFN-&#945; subtypes, is limited by their shared sequence identity; likewise, the sequences of the type III IFN, especially IFN-&#955;2 and -&#955;3, are highly similar. This assay provides a reliable, reproducible, and relatively inexpensive means to analyze the expression of the seventeen interferon subtype transcripts.

This protocol describes a high-throughput qRT-PCR assay for the analysis of type I and III IFN expression signatures. The assay discriminates single base pair differences between the highly similar transcripts of these genes. Through batch assembly and robotic pipetting, the assays are consistent and reproducible.

Yüksek verim Kantitatif Gerçek zamanlı RT-PCR Testi Türleri I ve III İnterferon Alttipleriyle İfadesi Profilleri belirlenmesi için

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

Use of a Monocyte Monolayer Assay to Evaluate Fc&#947; Receptor-mediated Phagocytosis

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly versatile in vitro assay that can be modified to examine different aspects of antibody and Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis in both research and clinical settings. The assay utilizes adherent monocytes from peripheral blood mononuclear cells isolated from mammalian whole blood. MMA has been described for use in both human and murine investigations. These monocytes express Fc&#947;Rs (e.g., Fc&#947;RI, Fc&#947;RIIA, Fc&#947;RIIB, and Fc&#947;RIIIA) that are involved in immune responses. The MMA exploits the mechanism of Fc&#947;R-mediated interactions, phagocytosis in particular, where antibody-sensitized red blood cells (RBCs) adhere to and/or activate Fc&#947;Rs and are subsequently phagocytosed by the monocytes. In vivo, primarily tissue macrophages found in the spleen and liver carry out Fc&#947;R-mediated phagocytosis of antibody-opsonized RBCs, causing extravascular hemolysis. By evaluating the level of phagocytosis using the MMA, different aspects of the in vivo Fc&#947;R-mediated process can be investigated. Some applications of the MMA include predicting the clinical relevance of allo- or autoantibodies in a transfusion setting, assessing candidate drugs that promote or inhibit phagocytosis, and combining the assay with fluorescent microscopy or traditional Western immunoblotting to investigate the downstream signaling effects of Fc&#947;R-engaging drugs or antibodies. Some limitations include the laboriousness of this technique, which takes a full day from start to finish, and the requirement of research ethics approval in order to work with mammalian blood. However, with diligence and adequate training, the MMA results can be obtained within a 24-h turnover time.

Use of a Monocyte Monolayer Assay to Evaluate Fc&amp;#947; Receptor-mediated Phagocytosis

The monocyte monolayer assay (MMA) is an in vitro assay that utilizes isolated primary monocytes obtained from mammalian peripheral whole blood to evaluate Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis.

Bir Monosit Tek tabakalı Testi Kullanımı Fcy reseptör aracılı fagositoz değerlendirin

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly ...

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 ...

A Simple Red Blood Cell Lysis Method for the Establishment of B Lymphoblastoid Cell Lines

A number of methods exist for the transformation of B lymphocytes by the Epstein Barr virus in vitro into immortalized cell lines. We have developed a new method with a powerful and simple strategy for the establishment of B-LCLs, the red blood cell lysis method. This method simplified the PBMC separation procedure with red blood cell removal, and used as little as 0.5 mL of whole blood for establishing EBV-immortalized cell lines, which can proliferate to large cell numbers in a relatively short amount time with a 100% success rate. The method is simple, reliable, time saving, and applicable to treating a large number of the clinical samples.

A simple red blood cell lysis method for establishing B-LCLs was developed with high immortalization efficiency, requiring a small amount of blood and saving time from initiation to cryopreservation.

B lenfoblastoid hücre dizilerinde Kurulması için Basit Kırmızı Kan Hücresi Lizis Yöntemi

A number of methods exist for the transformation of B lymphocytes by the Epstein Barr virus in vitro into immortalized cell lines. We have developed a new ...

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol specifically intended to measure antigen-specific killing without an infection. This protocol is designed and describes methods to overcome these limitations by allowing for the detection of antigen-specific killing of a target cell by CD8+ T cells in vivo. This is accomplished by merging a vaccination model with a traditional CFSE-labeled target killing assay. This combination allows the researcher to assess the antigen-specific CTL potential directly and quickly as the assay is not dependent upon tumor growth or infection. In addition, the readout is based on flow cytometry and so should be readily accessible to most researchers. The major limitation of the study is identifying the timeline in vivo that is appropriate to the hypothesis being tested. Variations in antigen strength and mutations in the T cells that may result in differential cytolytic function need to be carefully assessed to determine the optimal time for cell harvest and assessment. The appropriate concentration of peptide for vaccination has been optimized for hgp10025-33 and OVA257-264, but further validation would be needed for other peptides that may be more appropriate to a given study. Overall, this protocol allows a quick assessment of killing function in vivo and can be adapted to any given antigen.

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

The goal of this protocol is to allow for detection of in vivo antigen-specific killing of a target cell in a murine model.

Vivo Tahlil antijen spesifik T-hücre Cytolytic işlevi bir aşı modeli kullanılarak tespiti için

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol ...

Measuring Deformability and Red Cell Heterogeneity in Blood by Ektacytometry

Decreased red cell deformability is characteristic of several disorders. In some cases, the extent of defective deformability can predict severity of disease or occurrence of serious complications. Ektacytometry uses laser diffraction viscometry to measure the deformability of red blood cells subject to either increasing shear stress or an osmotic gradient at a constant value of applied shear stress. However, direct deformability measurements are difficult to interpret when measuring heterogenous blood that is characterized by the presence of both rigid and deformable red cells. This is due to the inability of rigid cells to properly align in response to shear stress and results in a distorted diffraction pattern marked by an exaggerated decrease in apparent deformability. Measurement of the degree of distortion provides an indicator of the heterogeneity of the erythrocytes in blood. In sickle cell anemia, this is correlated with the percentage of rigid cells, which reflects the hemoglobin concentration and hemoglobin composition of the erythrocytes. In addition to measuring deformability, osmotic gradient ektacytometry provides information about the osmotic fragility and hydration status of erythrocytes. These parameters also reflect the hemoglobin composition of red blood cells from sickle cell patients. Ektacytometry measures deformability in populations of red cells and does not, therefore, provide information on the deformability or mechanical properties of individual erythrocytes. Regardless, the goal of the techniques described herein is to provide a convenient and reliable method for measuring the deformability and cellular heterogeneity of blood. These techniques may be useful for monitoring temporal changes, as well as disease progression and response to therapeutic intervention in several disorders. Sickle cell anemia is one well-characterized example. Other potential disorders where measurements of red cell deformability and/or heterogeneity are of interest include blood storage, diabetes, Plasmodium infection, iron deficiency, and the hemolytic anemias due to membrane defects.

Here we present techniques to measure red cell deformability and cellular heterogeneity by ektacytometry. These techniques are applicable to general investigations of red cell deformability and specific investigations of blood diseases characterized by the presence of both rigid and deformable red cells in circulation, such as sickle cell anemia.

Deformasyon ve kan Ektacytometry tarafından kırmızı hücre heterojenite ölçme

Decreased red cell deformability is characteristic of several disorders. In some cases, the extent of defective deformability can predict severity of ...