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Platelet purification protocol

Yu Cheng, Xiao Dong Tony Wang, Stephan Jaenicke, Gaik-Khuan Chuah
Highly crystalline γ-zirconium phosphate has been synthesized by a novel minimalistic approach and investigated as a selective ion exchanger for cesium, ammonium and potassium. In contrast to current solution-based preparations, the mechanochemistry-based synthesis provides easy access to γ-zirconium phosphate with short synthesis times and low crystallization temperature. The addition of NaF as a mineralizer increases the crystallinity of γ-zirconium phosphate, which forms micrometer-sized uniformly shaped rectangular platelets...
March 23, 2018: Inorganic Chemistry
Pierre Bohec, Jérémie Gachelin, Véronique Ollivier, Thibaut Mutin, Xavier Télot, Benoît Ho-Tin-Noé, Sandra Sanfilippo
Purity, limited platelet activation, and preservation of platelet function are important stakes of preparation of platelet concentrates (PC) for clinical use. In fact, contaminating red blood cells and leukocytes, as well as activated and/or poorly functional platelets in PC, represents a risk of poor efficiency and adverse side effects during platelet transfusion. Therefore, optimization of preparation and storage of PC is still an active field of research. Shear-induced platelet activation is an unwanted side effect of the hard-spin (up to 5000g) step of centrifugation-based methods currently used in blood banks to prepare PC from whole blood samples...
December 6, 2017: Platelets
Stefanie Mak, Ruoyu Sun, Michael Schmalenberg, Carsten Peters, Peter B Luppa
Analysis of membrane proteins is still inadequately represented in diagnostics despite their importance as drug targets and biomarkers. One main reason is the difficult handling caused by their insolubility in aqueous buffer solutions. The nanodisc technology was developed to circumvent this challenge and enables analysis of membrane proteins with standard research methods. However, existing nanodisc generation protocols rely on time-consuming membrane isolation and protein purification from overexpression systems...
April 4, 2017: Biochemical Journal
Karin Pachler, Thomas Lener, Doris Streif, Zsuzsanna A Dunai, Alexandre Desgeorges, Martina Feichtner, Michaela Öller, Katharina Schallmoser, Eva Rohde, Mario Gimona
BACKGROUND AIMS: Extracellular vesicles (EVs) released by mesenchymal stromal cells (MSCs) may contribute to biological processes such as tissue regeneration, immunomodulation and neuroprotection. Evaluation of their therapeutic potential and application in future clinical trials demands thorough characterization of EV content and production under defined medium conditions, devoid of xenogenic substances and serum-derived vesicles. Addressing the apparent need for such a growth medium, we have developed a medium formulation based on pooled human platelet lysate (pHPL), free from animal-derived xenogenic additives and depleted of EVs...
April 2017: Cytotherapy
Paola Vinci, Antonio Bastone, Silvia Schiarea, Claudia Cappuzzello, Annalisa Del Prete, Erica Dander, Andrea Biondi, Giovanna D'Amico
BACKGROUND: Mesenchymal stromal cells (MSCs) are multipotent cells characterized by broad immunomodulatory properties exploited for the treatment of inflammatory disorders. However, the efficacy of MSC-based therapy is highly variable and tightly linked to MSC culture conditions and treatment schedule. Thus, the identification of novel key molecules regulating MSC immunomodulatory activities in vivo might constitute a crucial step toward the optimization of currently available clinical protocols...
February 2017: Cytotherapy
Basma HadjKacem, Héla Mkaouar, Ikram Ben Amor, Jalel Gargouri, Ali Gargouri
Glycocalicin (GC) is a large extracellular proteolytic fragment of glycoprotein Ib, a membrane platelet component playing an essential role in the physiological processes of platelet adhesion and aggregation. GC contains the binding sites for thrombin and von Willebrand factor. GC circulates normally in vivo in significant concentrations and the plasma level of this protein reflects a complex function of factors including platelet count or platelet turnover. It can therefore serve as a good indicator for many diseases like hypoplastic thrombocytopenia and idiopathic thrombocytopenic purpura...
January 1, 2016: Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences
Olivier Bausset, Jeremy Magalon, Laurent Giraudo, Marie-Laure Louis, Nicolas Serratrice, Corrine Frere, Guy Magalon, Françoise Dignat-George, Florence Sabatier
UNLABELLED: The platelet-rich plasma (PRP) is an autologous biotherapy commonly used for its healing properties. Once activated, platelets released a real "cocktail" of growth factor and cytokines implied in numerous regenerative processes. However the impact of medical practices associated to PRP therapeutic use on platelets functionality remains poorly known. OBJECTIVES: we evaluated the in vitro effects of two commonly used local anesthetics (Xylocaine(*) and Naropin(*)) on PRP functionality...
January 2014: Muscles, Ligaments and Tendons Journal
Lu Zhou, Fabien Sohet, Richard Daneman
This protocol describes the use of immunopanning to purify rodent pericytes from the optic nerve. Immunopanning permits the prospective isolation of pericytes from optic nerve tissue by relying on the binding of pericytes to an anti-PDGFRβ (platelet-derived growth factor receptor beta) antibody adhered to a Petri dish. The cells are viable at the end of this gentle procedure, and they can be analyzed acutely for gene expression or cultured alone or in coculture with other central nervous system (CNS) cell types, including CNS endothelial cells and CNS astrocytes...
June 2014: Cold Spring Harbor Protocols
Luke A Yates, Robert J C Gilbert
Kindlins are essential coactivators, with talin, of the cell surface receptors integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin β-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption...
March 19, 2014: Journal of Visualized Experiments: JoVE
Shauna A Trichler, Sandra C Bulla, John Thomason, Kari V Lunsford, Camilo Bulla
BACKGROUND: Several research applications involving platelets, such as proteomic and transcriptomic analysis, require samples with very low numbers of contaminating leukocytes, which have considerably higher RNA and protein content than platelets. We sought to develop a platelet purification protocol that would minimize contamination, involve minimal centrifugation steps, and yield highly pure platelet samples derived from low volume whole blood samples from healthy dogs. RESULTS: Using an optimized OptiPrep density gradient technique, platelet recovery was 51...
2013: BMC Veterinary Research
Olivier Bausset, Laurent Giraudo, Julie Veran, Jeremy Magalon, Jean-Marie Coudreuse, Guy Magalon, Christophe Dubois, Nicolas Serratrice, Françoise Dignat-George, Florence Sabatier
The platelet-rich plasma (PRP) is an autologous biotherapy based on platelet-healing properties. Here, we developed a simple and reproducible PRP purification protocol based on two successive centrifugations. We evaluated different centrifugation speeds and time-storage durations on the platelet quantity and quality. Sterility and stability of our PRP homemade product were also performed. We prepared PRP from 54 healthy volunteers. We tested activation state, reactivity, and stability of platelets by flow cytometry using basal and adenosine diphosphate (ADP)-induced P-selectin expression markers; growth factor release after platelet activation by an enzyme-linked immunosorbent assay (ELISA); platelet aggregation capacity by aggregrometry assays; clot formation and retraction by thromboelastography; and platelet morphology by ultrastructural analysis...
June 2012: BioResearch Open Access
Hao Huang, Hans J Vogel
The Calcium- and Integrin-Binding protein 1 (CIB1) has been identified as an important regulatory Ca(2+)-binding protein that is involved in various cellular functions. Nuclear Magnetic Resonance (NMR) spectroscopy provides a powerful approach to study the structure, dynamics, and interactions of CIB1 and related proteins. Multidimensional NMR spectroscopy combined with various selective isotope labeling strategies has proven to be successful in the structure determination of CIB1. Moreover, the same approach allowed the detection of conformational changes when the protein binds different metal ions, and it facilitated the study of the interaction of CIB1 with the cytoplasmic domain of the human integrin αIIb subunit...
2013: Methods in Molecular Biology
Franziska T Arlinghaus, Johannes A Eble
Many snake venoms are known for their antithrombotic activity. They contain components that specifically target different platelet-activating receptors such as the collagen-binding integrin α2β1 and the von Willebrand factor receptor GPIb. In a search for an α2β1 integrin-blocking component from the venom of the habu snake (Trimeresurus flavoviridis), we employed two independent purification protocols. First, we used the integrin α2A domain, a major collagen-binding domain, as bait for affinity purification of an α2β1 integrin-binding toxin from the crude venom...
January 11, 2013: Journal of Biological Chemistry
Chy-Anh Tran, Monica Torres-Coronado, Agnes Gardner, Angel Gu, Hieu Vu, Anitha Rao, Lan-Feng Cao, Amira Ahmed, David Digiusto
Cell separation by counterflow centrifugal elutriation has been described for the preparation of monocytes for vaccine applications, but its use in other current good manufacturing practice (cGMP) operations has been limited. In this study, growth factor-mobilized peripheral blood progenitor cell products were collected from healthy donors and processed by elutriation using a commercial cell washing device. Fractions were collected for each product as per the manufacturer's instructions or using a modified protocol developed in our laboratory...
May 2012: Stem Cells Translational Medicine
Hidetoshi Sakurai, Yasuko Sakaguchi, Emi Shoji, Tokiko Nishino, Izumi Maki, Hiroshi Sakai, Kazunori Hanaoka, Akira Kakizuka, Atsuko Sehara-Fujisawa
Induced pluripotent stem (iPS) cells are generated from adult somatic cells by transduction of defined factors. Given their unlimited proliferation and differentiation potential, iPS cells represent promising sources for cell therapy and tools for research and drug discovery. However, systems for the directional differentiation of iPS cells toward paraxial mesodermal lineages have not been reported. In the present study, we established a protocol for the differentiation of mouse iPS cells into paraxial mesodermal lineages in serum-free culture...
2012: PloS One
Mark P White, Abdul J Rufaihah, Lei Liu, Yohannes T Ghebremariam, Kathryn N Ivey, John P Cooke, Deepak Srivastava
Recent evidence suggests human embryonic stem cell (hESC) and induced pluripotent stem (iPS) cell lines have differences in their epigenetic marks and transcriptomes, yet the impact of these differences on subsequent terminally differentiated cells is less well understood. Comparison of purified, homogeneous populations of somatic cells derived from multiple independent human iPS and ES lines will be required to address this critical question. Here, we report a differentiation protocol based on embryonic development that consistently yields large numbers of endothelial cells (ECs) derived from multiple hESCs or iPS cells...
January 2013: Stem Cells
A Signore, A W J M Glaudemans, G Malviya, E Lazzeri, N Prandini, A L Viglietti, E F J De Vries, R A J O Dierckx
AIM: White blood cell (WBC) labelling requires isolation of cells from patient's blood under sterile conditions using sterile materials, buffers and disposables under good manufacturing practice (GMP) conditions. Till now, this limited the use of white blood cell scintigraphy (WBC-S) only to well equipped laboratories with trained personnel. We invented, developed and tested a disposable, sterile, closed device for blood manipulation, WBC purification and radionuclide labelling without exposing patient's blood and the operator to contamination risks...
August 2012: Quarterly Journal of Nuclear Medicine and Molecular Imaging
Katrin Watschinger, Ernst R Werner
Ether lipids are an emerging class of lipids which have so far not been investigated and understood in every detail. They have important roles as membrane components of e.g. lens, brain and testis, and as mediators such as platelet-activating factor. The metabolic enzymes for biosynthesis and degradation have been investigated to some extent. As most involved enzymes are integral membrane proteins they are tricky to handle in biochemical protocols. The sequence of some ether lipid metabolising enzymes has only recently been reported and other sequences still remain obscure...
January 2013: Biochimie
C Lim, D P Manage, A Atrazhev, G Denomme, C J Backhouse, J P Acker
Centralised laboratories routinely determine blood types by serological and molecular methods. Current practices have limitations in terms of cost, time and accessibility. Miniaturised microfluidic platforms offer an alternative to conventional genotyping methods, since they consume fewer reagents, provide faster analysis and allow for complete integration and automation. As these 'lab-on-a-chip' devices have been used for bacterial and viral detection, the authors investigated blood group genotyping as a novel application of microfluidic technology...
June 2012: IET Nanobiotechnology
Lenka Sadilkova, Zoltan Paluch, Jirina Mottlova, Frantisek Bednar, Stefan Alusik
BACKGROUND: Thromboxane B2 (TxB2) and particularly 11-dehydrothromboxane B2 (11-dTxB2) are widely used as prognostic risk markers of platelet activation in cardiovascular diseases. The main errors in TxB2 and 11-dTxB2 determination include either low concentrations of circulating TxB2 (1 - 2 pg/mL) and 11-dTxB2 (0.9 - 4.3 pg/mL) or rather high transiency (mean TxB2 half-life is approximately 5 minutes) as well as an incorrect pre-analytical phase set up. The aim of this study was to investigate the impact of a widely used purification step on the results of enzyme immunosorbent assay (EIA)--based measurement of the two selected thromboxanes...
2012: Clinical Laboratory
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