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Platelet purification protocol

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https://www.readbyqxmd.com/read/26606109/purification-of-glycocalicin-from-human-plasma
#1
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
https://www.readbyqxmd.com/read/24932442/impact-of-local-anaesthetics-and-needle-calibres-used-for-painless-prp-injections-on-platelet-functionality
#2
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
https://www.readbyqxmd.com/read/24890207/purification-of-pericytes-from-rodent-optic-nerve-by-immunopanning
#3
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
https://www.readbyqxmd.com/read/24686835/efficient-production-and-purification-of-recombinant-murine-kindlin-3-from-insect-cells-for-biophysical-studies
#4
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
https://www.readbyqxmd.com/read/23866028/ultra-pure-platelet-isolation-from-canine-whole-blood
#5
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
https://www.readbyqxmd.com/read/23516671/formulation-and-storage-of-platelet-rich-plasma-homemade-product
#6
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
https://www.readbyqxmd.com/read/23296607/purification-and-stable-isotope-labeling-of-the-calcium-and-integrin-binding-protein-1-for-structural-and-functional-nmr-studies
#7
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
https://www.readbyqxmd.com/read/23204528/the-collagen-binding-integrin-%C3%AE-2%C3%AE-1-is-a-novel-interaction-partner-of-the-trimeresurus-flavoviridis-venom-protein-flavocetin-a
#8
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
https://www.readbyqxmd.com/read/23197821/optimized-processing-of-growth-factor-mobilized-peripheral-blood-cd34-products-by-counterflow-centrifugal-elutriation
#9
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
https://www.readbyqxmd.com/read/23115636/in-vitro-modeling-of-paraxial-mesodermal-progenitors-derived-from-induced-pluripotent-stem-cells
#10
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
https://www.readbyqxmd.com/read/23079999/limited-gene-expression-variation-in-human-embryonic-stem-cell-and-induced-pluripotent-stem-cell-derived-endothelial-cells
#11
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
https://www.readbyqxmd.com/read/23013669/development-and-testing-of-a-new-disposable-sterile-device-for-labelling-white-blood-cells
#12
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
https://www.readbyqxmd.com/read/22771767/orphan-enzymes-in-ether-lipid-metabolism
#13
REVIEW
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
https://www.readbyqxmd.com/read/22559704/microfluidic-approach-to-genotyping-human-platelet-antigens
#14
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
https://www.readbyqxmd.com/read/22372363/the-purification-step-is-not-crucial-in-eia-measurements-of-thromboxane-b2-and-11-dehydrothromboxane-b2-in-human-plasma
#15
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
https://www.readbyqxmd.com/read/22130711/megakaryocyte-and-platelet-production-from-human-cord-blood-stem-cells
#16
Amélie Robert, Valérie Cortin, Alain Garnier, Nicolas Pineault
The cloning of thrombopoietin together with advances in the culture of hematopoietic stem cells have paved the way for the study of megakaryopoiesis, ongoing clinical trials and, in the future, for the potential therapeutic use of ex vivo produced blood substitutes, such as platelets. This chapter describes a 14-day culture protocol for the production of human megakaryocytes (MKs) and platelets, and assays that can be used to characterize the functional properties of the platelets produced ex vivo. CD34(+) cells isolated from cord blood cells are grown in a serum-free medium supplemented with newly developed cytokine cocktails optimized for MK differentiation, expansion, and maturation...
2012: Methods in Molecular Biology
https://www.readbyqxmd.com/read/22130707/a-rapid-and-efficient-platelet-purification-protocol-for-platelet-gene-expression-studies
#17
Stefan Amisten
Isolation of pure platelet samples from whole blood is crucial for the study of platelet gene expression. The main obstacles to overcome in order to successfully isolate platelets from whole blood include (1) platelet activation; (2) leukocyte and red blood cell contamination, and (3) time-dependent platelet mRNA degradation. This chapter describes a rapid and highly efficient method for isolating human circulating platelets from small volumes of whole blood based on efficient inhibition of platelet activation and leukocyte removal by filtration followed by magnetic bead-depletion of residual contaminating leukocytes and red blood cells...
2012: Methods in Molecular Biology
https://www.readbyqxmd.com/read/21468956/phosphoproteome-analysis-of-the-platelet-plasma-membrane
#18
Thomas Premsler, Urs Lewandrowski, Albert Sickmann, René Peiman Zahedi
Blood platelets are key players standing at the crossroads between physiologically occurring hemostasis and pathologic thrombus formation. As these cellular particles lack a nucleus, intra- and intercellular processes involved in platelet activity and function are almost exclusively regulated on the protein level. In particular, posttranslational protein modification by phosphorylation, which allows for a quick and highly dynamic transduction of cellular signals, is discussed in this context. In addition, since platelet activation and aggregation usually require surface contact with the surrounding tissue, special interest focuses on this contacting region, and hence on the subproteome of the platelet plasma membrane...
2011: Methods in Molecular Biology
https://www.readbyqxmd.com/read/20431540/synthesis-of-alkyl-and-aryl-amino-substituted-anthraquinone-derivatives-by-microwave-assisted-copper-0-catalyzed-ullmann-coupling-reactions
#19
Younis Baqi, Christa E Müller
This protocol describes the efficient, generally applicable Ullmann coupling reaction of bromaminic acid with alkyl- or aryl-amines in phosphate buffer under microwave irradiation using elemental copper as a catalyst. The reaction leads to a number of biologically active compounds. As a prototypical example, the synthesis of a new, potent antagonist of human platelet P2Y(12) receptors, which has potential as an antithrombotic drug, is described in detail. The optimized protocol includes a description of an appropriate reaction setup, thin layer chromatography for monitoring the reaction and a procedure for the isolation, purification and characterization of the anticipated product...
May 2010: Nature Protocols
https://www.readbyqxmd.com/read/20235104/isolation-of-platelet-granules
#20
Juliane Niessen, Gabriele Jedlitschky, Andreas Greinacher, Heyo K Kroemer
Functional analysis of platelet intracellular structures requires isolation and purification of these cellular compartments. With regard to the function of platelets, both, dense (delta) and alpha granules are relevant target structures. However, the availability of sufficient purification protocols for these structures is rather limited. This unit describes two protocols for isolation and purification of platelet granule structures. The Basic Protocol describes a new technique based on immunolabeling with target-specific antibodies followed by magnetic sorting, whereas the Alternate Protocol describes the more traditional procedure based on differential centrifugation and density-based sedimentation...
March 2010: Current Protocols in Cell Biology
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