Gli autori riconoscono il finanziamento attraverso il Dipartimento della Difesa dal Congresso Regia programmi di ricerca medica (# W81XWH-10-1-0445), National Institutes of Health (NIH R21 HL110056), e American Heart Association (# 11SDG4890030).

1. Preparazione e sterilizzazione di buffer e agenti di test <ol><li> 150 mM NaCl tampone: sciogliere 4,383 g di cristalli di NaCl in 500 ml di acqua Nanopure. </li><li> Buffer pH: Preparare tamponi fosfato a pH 5.6, 6.2, 6.8, e 7.4 mescolando quantità appropriate di monobasico e sodio fosfato bibasico. Se i campioni devono essere sottoposti a valori di pH più bassi (pH &lt;5.6) poi un buffer più appropriato, come il tampone citrato, dovrebbe essere usato. Ricette del buffer sono facilmente disponibili, e un esempio di riferimento è stato fornito qui 12. </li><li> Sterilizzare tutti i buffer notato in precedenza attraverso una bottiglia-top gli aspiratori per filtrazione e ricontrollare tampone di pH. </li><li> 20% Triton X-100 (controllo positivo): Mescolare 20 ml puro Triton X-100 in 80 ml di acqua Nanopure. Vortex vigorosamente e ultrasuoni per sciogliere. Lasciare a temperatura ambiente per una notte prima dell&#39;uso. </li></ol> 2. Preparazione di eritrociti <ol><li> Ottenere 25 ml di sangue from un donatore anonimo umano, disegnato direttamente in K 2-EDTA rivestite vacutainer per prevenire la coagulazione. </li></ol> NOTA: Tutte le procedure devono essere pre-approvato dal Consiglio appropriata revisione istituzionale (IRB) e prelievo venoso e raccolta del sangue deve essere eseguita da un flebotomo addestrato al fine di minimizzare il rischio per il donatore. Procedure standard di flebotomia sono stati pubblicati altrove 13. <ol start="2"><li> Centrifugare il sangue a 500 xg per 5 minuti, ed i livelli di ematocrito marchio (rosso, strato inferiore) e plasma (giallo, strato superiore) sul tubo. </li><li> Aspirare delicatamente plasma tramite una micropipetta, aggiungere in candeggina, e gettare in rifiuti a rischio biologico. </li><li> Riempire tubo di ematocrito alla linea marcata (livello iniziale del plasma) con 150 mM di soluzione di NaCl. Chiudere e capovolgere un paio di volte per mescolare delicatamente. Centrifugare a 500 xg per 5 min. </li><li> Ripetere il passaggio 2,3-2,4 per lavare le cellule del sangue di nuovo. Poi aspirare Supernatformica e sostituirlo con PBS a pH 7,4. Capovolgere per miscelare. </li><li> Dividere il sangue in modo uniforme in quattro tubi, corrispondente a ciascun pH che sarà testato. Etichettare i tubi secondo ognuna pH da testare (5,6, 6,2, 6,8, 7,4). </li><li> Centrifugare provette a 500 xg per 5 min. Segna i livelli su tubi, quindi aspirare il surnatante. </li><li> Riempire ogni provetta di linea contrassegnata con tampone di pH adeguato (come indicato in 2.6). </li><li> Etichettare quattro provette da 50 ml coniche (uno per pH a test), e pipettare 49 ml di PBS a pH appropriato in ciascuna provetta conica. </li><li> Aggiungere 1 ml di eritrociti (pH stesso) nel tubo corrispondente per una diluizione 1:50. Ispezionare visivamente il sangue diluito, che deve essere torbida e si sistemerà, se lasciati indisturbati. Se nessuna forma di pellet, le cellule sono lisate. </li></ol> 3. Lysis Assay 96 pozzetti Piastra di supporto e di quantificazione <ol><li> Preparare soluzioni di base di tutti gli agenti sperimentali di consegna della droga, a 20 volte la concentrazione finale desiderata da sottoporre a prova (testavrà 10 ml di agente di consegna della droga + 190 ul cellule rosse del sangue diluito, che portano ad una diluizione 1/20 di un agente originale di somministrazione di farmaci in miscela di prova finale). Stock di 20, 100, e 800 pg / ml sono suggeriti, risultante in concentrazioni finali di 1, 5, e 40 pg / ml, rispettivamente. </li><li> Pipettare 10 ml di ciascuna soluzione madre in fondo a V da 96 pozzetti. Per ottenere risultati ottimali, caricare ciascun campione in triplicato o quadruplicato. </li></ol> NOTA: per facilità, si raccomanda una distinta piastra a 96 pozzetti deve essere preparato per ciascun pH da testare, con ciascun campione (per ogni concentrazione) caricato con n = 3-4 per piastra. <ol start="3"><li> Per pozzetti di controllo positivi, aggiungere 10 ml di 20% Triton X-100. </li><li> Per i pozzetti di controllo negativo, aggiungere 10 ml di tampone fosfato. Utilizzare tampone al pH stesso da testare. </li><li> Dispensare 190 ml di eritrociti diluiti (vedi 2.10) in ogni pozzetto, assicurandosi soluzione madre cella rimaneomogenea durante il trasferimento. Suggerimento: Utilizzare pipetta multicanale per semplificare questo compito. </li><li> Incubare le piastre a 37 ° C per un&#39;ora (Opzionale: Utilizzare un agitatore orbitale o rocker). </li><li> Centrifugare piastre per 5 min a 500 xg per eritrociti pellet intatti. Nota: quando si rimuove la placca dalla centrifuga e il trasporto al passo successivo, maneggiare con cura ed essere certi di non disturbare il pellet. </li><li> Usando una pipetta multicanale, trasferire 100 pl di surnatante da ciascun pozzetto in una chiara, a fondo piatto da 96 pozzetti. Nota: se il pellet cellulare è accidentalmente disturbato per ogni campione (s), si può ri-centrifugare la piastra e quindi procedere con il trasferimento supernatante. </li><li> Misurare assorbanza di supernatanti con un lettore di piastre. Si noti che un intervallo di lunghezze d&#39;onda possono essere utilizzati (400-541 nm). </li></ol> NOTA: lettori di piastre diverse possono avere punti di saturazione e sensibilità diverse, in modo che la scelta di una lunghezza d&#39;onda per measurements dipende se i dati di emolisi campioni sperimentali può essere normalizzati affidabile contro emolisi massima indotta dal trattamento detergente. Questo richiede una misurazione precisa valori di assorbanza dei campioni di controllo positivi. <ol start="10"><li> Utilizzo di Microsoft Excel o un simile software di analisi dei dati, trovare la media delle letture di assorbanza lo sfondo da campioni di controllo negativo create per ogni pH (punto 3.4). Sottrarre questo valore assorbanza di fondo da tutti gli altri campioni che sono stati misurati in quel pH. </li><li> Dopo sottrazione del fondo, trovare l&#39;assorbanza media del controllo positivo detergente trattati con campioni (punto 3.3). Poi normalizzare tutti i punti dati sperimentali a questo valore medio di assorbanza, che dovrebbe rappresentare il 100% emolisi. Infine, moltiplicare ogni valore ben del 100% per calcolare emolisi% che si è verificato in ogni singolo pozzetto rispetto al controllo detergente. </li></ol>

<ol>
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IRB di approvazione: Procedure che coinvolgono soggetti umani sono stati approvati dal Comitato di Revisione Vanderbilt University Consiglio Istituzionale (IRB; protocollo # 111251). Nessun conflitto di interessi dichiarati.

pH-sensibili polimeri o altri agenti destinati endosomolytic funzione può essere rapidamente ed efficacemente proiettato sulla base di lisi dei globuli rossi a pH incontrate in endosomi (Figura 1; pH 6,8 - endosoma precoce, pH 6,2 - endosoma tardi, pH 5,6 - lisosoma). 14-17 pH-dipendente emolisi è stato utilizzato per schermare la capacità dei vettori di mediare endosomal rilascio di peptidi terapeutici biomacromolecular (ad esempio, siRNA, ODN, proteine), ed i risultati di questa...

Sebbene vi siano molti potenziali obiettivi terapeutici impatto elevato all&#39;interno della cellula, la consegna intracellulare di agenti rappresenta una sfida significativa. Spesso, i farmaci biologici, in particolare, vengono internalizzati dalle cellule e trafficata in vescicole che o portare al degrado del loro contenuto tramite l&#39;endo-lisosomiale via, o vengono trasportati indietro fuori dalla cellula tramite esocitosi. 1 In quest&#39;ultimo processo, il pH interno delle vescicole viene acidificata a circa 5-6, che è il pH ottimale per l&#39;attività di enzimi che funzionano in questo vano, ad esempio il lisozima. 2 Recentemente, un certo numero di materiali sono stati specificamente progettati per sfruttare l&#39;acidificazione di endosomi per facilitare l&#39;erogazione citosolico del loro carico. Un esempio di questo approccio utilizza nanoparticelle polimeriche sintetiche, micelle cui anima è zwitterionici e carica neutra a pH fisiologico (cioè 7,4). Tuttavia, a pH 6,0- 6,5, i polimeri diventano protonati e acquisire una carica positiva netta che destabilizza il nucleo micelle, e segmenti polimerici esposti interagire e distruggere la membrana endosomiale. Questa attività è stato dimostrato per promuovere la fuga endosomiale di peptidi e acido nucleico-terapie basate, consentendo loro di accedere ai bersagli citosolico. 3,4 Altri esempi di metodi sviluppati per mediare fuga endosomiale che distruggere la barriera membrana includono &#39;fusogenico&#39; peptidi o proteine ​​che possono mediare la fusione della membrana o transitoria formazione di pori nel doppio strato fosfolipidico. 5 omopolimeri di acidi acrilici anionici alchile come acido poli (propylacrylic) sono un altro approccio ben studiato, e in questi polimeri, lo stato di protonazione di acido carbossilico detta sospensione transizione in una idrofobica, membrana dirompente stato in endo-lisosomiale intervalli di pH. 6,7 Un sistema modello utile per lo screening di comportamento endosomolytic è l&#39;indirizzox pH-dipendente vivo dosaggio emolisi. 8 In questo sistema modello, la membrana eritrocitaria serve come un surrogato della membrana a doppio strato lipidico che racchiudono endo-vescicole lisosomiali. Questo modello generalizzabile è stato utilizzato da altri per valutare il comportamento endosomolytic di penetrazione cellulare peptidi e altri sistemi polimerici consegna del gene. 8-11 In questo esperimento, globuli rossi umani e materiali di prova sono co-incubate in tampone a pH definiti che mimano extracellulari (7.4), precoce endosomali (6.8), e la fine del endo-lisosomiale (&lt;6.8) ambienti. La quantità di emoglobina rilasciata durante il periodo di incubazione è quantificato come misura di lisi dei globuli rossi, che viene normalizzato alla quantità di emoglobina rilasciata in campioni di controllo positivi lisate con un detergente. Da screening di una piccola libreria di materiali di prova potenzialmente endosomolytic, si può dedurre che i campioni che non producono emolisi a pH 7.4, ma orlo significativamente elevatiolysis a pH &lt;6.5, saranno i candidati più efficaci e cytocompatible per la consegna della droga citosolico. Materiali che soddisfano questi criteri dovrebbe rimanere inerte e non indiscriminatamente distruggere le membrane a doppio strato lipidico (cioè che potrebbe causare citotossicità) fino ad essere esposto ad una diminuzione del pH locale dopo interiorizzazione in endo-compartimenti lisosomiali. In questo protocollo, eritrociti sono isolati da un donatore umano e co-incubate a pH 5,6, 6,2, 6,8, 7,4 o con agenti farmaci sperimentali endosomolytic consegna. Eritrociti intatti sono pellettizzati, ei surnatanti (contenenti emoglobina rilasciata da eritrociti lisati) vengono analizzati per l&#39;assorbanza caratteristica di emoglobina mediante un lettore di piastre (Figura 1).

<table border="1"><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti (opzionale) </td></tr><tr><td> BD Vacutainer - K2EDTA Vacutainer Tubi </td><td> Fisher Scientific </td><td> 22-253-145 </td><td> Per la raccolta del sangue </td></tr><tr><td> BD Vacutainer sangue aghi di raccolta, 20,5 scartamento </td><td> Fisher Scientific </td><td> 02-665-31 </td><td> Per la raccolta del sangue </td></tr><tr><td> BD Vacutainer tubo porta / ago Adattatore </td><td> Fisher Scientific </td><td> 22-289-953 </td><td> Per la raccolta del sangue </td></tr><tr><td> BD Marca isopropilico tamponi imbevuti di alcool </td><td> Fisher Scientific </td><td> 13-680-63 </td><td> Per la raccolta del sangue </td></tr><tr><td> BD Vacutainer Latex-Free Tourniquet </td><td> Fisher Scientific </td><td> 02-657-6 </td><td> Per la raccolta del sangue </td&gt; </tr><tr><td> Acido cloridrico (conc.) </td><td> Fisher Scientific </td><td> A144-500 </td><td> Per la regolazione del pH del D-PBS. </td></tr><tr><td> Triton X-100 </td><td> Sigma-Aldrich </td><td> T8787 </td><td> Controllo positivo </td></tr><tr><td> Dulbecco PBS </td><td> Invitrogen </td><td> 14190 </td><td></td></tr><tr><td> Nalgene MF75 sterile monouso Bottle-Top Unità Filtro con membrana SFCA </td><td> Fisher Scientific </td><td> 09-740-44A </td><td></td></tr><tr><td> BD piastre a 96 pozzetti, a fondo piatto, di coltura trattata con polistirolo </td><td> Fisher Scientific </td><td> 08-772-2C </td><td> Per piastra di lettura al termine del saggio. </td></tr><tr><td> BD piastre a 96 pozzetti, fondo tondo, di coltura trattata con polistirolo </td><td> Fisher Scientific </td><td> 08-772-17 </td><td> Per incubazione di globuli rossi con agenti sperimentali. </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

Doppi strati di fosfolipidi che costituiscono endo-lisosomiale vescicole possono rappresentare un ostacolo alla consegna di farmaci biologici a bersagli intracellulari. Per superare questo ostacolo, una serie di trasportatori di farmaci sintetici sono stati progettati per interrompere attivamente la membrana endosomiale e consegnare cargo nel citoplasma. Qui, descriviamo il saggio emolisi, che può essere usato come un rapido, ad alta resa per la schermata citocompatibilità e attività endosomolytic dei sistemi intracellulari drug delivery. Nel saggio emolisi, globuli rossi umani e materiali di prova sono co-incubate in tampone a pH definiti che imitano extracellulari, endosomali precoce e tardiva endo-lisosomiali ambienti. A seguito di una fase di centrifugazione a pellet intatti cellule rosse del sangue, la quantità di emoglobina rilasciata nel terreno viene misurata spettrofotometricamente (405 nm per migliore dinamica). Il rosso per cento rottura delle cellule del sangue viene poi quantificato rispetto al controllo positivo samples lisate con un detergente. In questo sistema modello la membrana eritrocitaria funge da surrogato per la membrana lipidica a due strati che racchiudono endo-lisosomiale vescicole. Il risultato desiderato è emolisi trascurabile a pH fisiologico (7,4) ed emolisi robusta in endo-lisosomiale intervallo di pH da circa pH 5-6,8.

<html> In genere, gli agenti che presentano ideale pH-dipendente comportamento emolitica hanno il più alto potenziale per la consegna citosolico di farmaci, acidi nucleici, o altre molecole bioattive. Questo è esemplificato da Agent # 1 come rappresentato in figura 2, che presenta emolisi minima a pH 7,4, ma un forte aumento comportamento emolitica a intervalli di pH endosomiali (&lt;6,5). Alcuni agenti possono esibire livelli significativi di comportamento emolitica a intervalli di pH fisiologici (Age...

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

Un saggio di emolisi può essere usato come un rapido, ad alta resa di schermo citocompatibilità sistemi di rilascio di farmaci e l&#39;attività endosomolytic per la consegna del carico intracellulare. Il saggio misura la rottura di membrane eritrocitarie in funzione del pH.

Ex Vivo Blood Red emolisi test cellulare per la valutazione degli agenti Endosomolytic pH-sensibile per la consegna dei farmaci citosolico Biomacromolecular

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

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Research

JoVE Journal

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 Views.  Vanderbilt University. Un saggio di emolisi può essere usato come un rapido, ad alta resa di schermo citocompatibilità sistemi di rilascio di farmaci e l'attività endosomolytic per la consegna del carico intracellulare. Il saggio misura la rottura di membrane eritrocitarie in funzione del pH.

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.

L&#39;introduzione di shear stress nello studio di adesione batterica

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

Ricerca

Immunologia e infezioni

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.

Una valutazione quantitativa della migrazione cellulare con il Test Phagokinetic motilità Traccia

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.

Gli antigeni Protetto funzionali globuli rossi dalla membrana innesto di Compact Poligliceroli Hyperbranched

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.

Metodi per quantitativa individuazione di anticorpi indotta da attivazione del complemento sui globuli rossi

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.

High-throughput Quantitative Real-time RT-PCR test per la determinazione di espressione Profili di tipo I e III interferone sottotipi

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.

L&#39;uso di un monociti monostrato test per valutare fagocitosi Fcy recettore-mediata

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.

Destrano migliora l'efficienza di trasduzione lentivirale di cellule NK primarie Murine ed umane

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.

Un cellulare semplice Red Blood Metodo Lysis per l&#39;istituzione di B linfoblastoidi linee cellulari

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.

In Vivo Test per la determinazione della funzione citolitica di cellule T antigene-specifiche utilizzando un modello di vaccinazione

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.

Misurazione di deformabilità e l'eterogeneità delle cellule rosse nel sangue di ektacitometria

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