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Reticulocytes

Reticulocytes are immature red blood cells that are transitional between the erythroblast and the mature erythrocyte.
They contain remnants of the nucleus and ribosomal RNA, giving them a reticulated appearance under the microscope.
Reticulocyte count is an important indicator of erythropoiesis and can provide insight into various hematological conditions such as anemia, hemolysis, and blood loss.
Studying reticulocyte dynamics can help clinicinas and researchers optimize research protocols and enhance reproducibility in their work.

Most cited protocols related to «Reticulocytes»

All transcripts utilized PCR products as templates for in vitro transcription (Sharma et al., 2010 (link)) except β-VHP-SL, which was transcribed from linearized plasmid. β-VHP-pA transcript was generated by treating β-VHP transcript with poly(A) polymerase to add an ∼200 nt poly(A) tail. 33P-labeled transcripts were generated with 33P-UTP (Perkin-Elmer) in transcription reactions. In vitro translation reactions using rabbit reticulocyte lysate (RRL), phenyl-depleted RRL, and DEAE fractionated RRL (Fr-RRL) were done as before (Sharma et al., 2010 (link); Hessa et al., 2011 (link)). ΔHbs1 RRL was generated by incubating 800 μl RRL with 200 μl of Pelota resin (immobilized via CnBr). Unconjugated and quenched CnBr resin served as a control. Unless indicated otherwise, translation reactions were for 60 min at 32°C. Where indicated, WT or DN Hbs1 was added at 10 min together with 100 μM aurin tricarboxylic acid to inhibit initiation. For direct analyses, translation reactions were denatured in 1% SDS and heated to 100°C. For downstream applications, translation reactions were cooled on ice and manipulated at 0°C–4°C for RNC isolation, sucrose gradients, and native immunoprecipitations (IPs).
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Publication 2013
2-diethylaminoethanol Aurintricarboxylic Acid Cardiac Arrest Cyanogen Bromide Immunoprecipitation isolation Plasmids Poly(A) Tail Polynucleotide Adenylyltransferase Rabbits Resins, Plant Reticulocytes Sucrose Transcription, Genetic
Constructs, antibodies, and recombinant proteins derived from earlier studies2 ,18 are fully described in Supplementary Methods. Antibodies to Bat3 and Ubl4A were produced in rabbits immunized with recombinant Bat3 fragment (residues 1-250) or full-length Ubl4A, respectively. TRC35 antibodies were against a synthetic peptide (NH2-CGSPIELD-COOH) conjugated to KLH. In vitro transcription, translation in reticulocyte lysate, sucrose gradient fractionation, chemical crosslinking, and immunoprecipitation was as before2 . RNCs were generated by transcription and translation of PCR products lacking a stop codon and sucrose gradient purified for use in the capture assay. CTAB precipitation of tRNA-associated polypeptides has been described28 .
Publication 2010
Antibodies Biological Assay Cetrimonium Bromide Codon, Terminator Fractionation, Chemical Immunoprecipitation Oryctolagus cuniculus Peptides Polypeptides Recombinant Proteins Reticulocytes Sucrose Transcription, Genetic Transfer RNA
Reticulocytes were isolated from 10 ml of peripheral blood in heparin following a process of leukodepletion [20 (link)]. In brief, the reticulocyte-enriched, leukocyte-depleted red cell eluate was centrifuged to recover the reticulocytes which were then resuspended in 2 ml of cold PBS and 5 ml of TRI reagent (T-9424, Sigma-Aldrich) and stored at -80°C until RNA extraction.
RNA extraction was carried out on the thawed samples according to the manufacturer's protocol. The precipitated RNA was resuspended in 50 μl DEPC water and 1 microgram of RNA was reverse transcribed with 200U SuperScript™ III Reverse Transcriptase (Invitrogen) for 60 min at 50°C using Oligo-dT in a 20 μl volume.
Purity of reticulocytes, assessed by a Sysmex automated blood cell analyser, was 1 leukocyte per 35.5 × 103 RBCs and no detectable platelets.
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Publication 2006
BLOOD Blood Cells Blood Platelets Cold Temperature Erythrocytes Heparin Leukocytes oligo (dT) Reticulocytes RNA-Directed DNA Polymerase
Wild-type and selenomethionyl proteins were expressed in E. coli and purified using Ni-NTA and size-exclusion chromatography. Crystals were grown in hanging drop, vapour diffusion format. Diffraction data were collected from cryo-protected crystals at beamline 21-IDG of the Advanced Photon Source (Argonne National Laboratories). The structure of S. cerevisiae Get3 complexed with ADP·AlF 4 was determined by single-wavelength anomalous dispersion (SAD) from selenomethionine-containing protein using PHENIX33 (link). The structures of nucleotide-free S. cerevisiae and S. pombe Get3 were solved by molecular replacement in PHASER34 (link). A monomer of the S. cerevisiae Get3–ADP·AlF 4 complex (with nucleotide and the α-helical subdomain removed) was used as the search model. Refinement and model building were carried out with PHENIX33 (link) and COOT35 (link).
A series of GET3 genes containing site-specific mutations were generated by Quik-Change mutagenesis. The identity of each mutant was confirmed by DNA sequencing. Proteins were expressed in E. coli and purified by Ni-NTA chromatography. TA substrate binding was monitored using a native pull-down assay in which full-length 35S-labelled human SEC61β was translated in a TRC40-depleted reticulocyte lysate translation extract with or without recombinant wild-type or mutant Get3 protein. After translation, Get3 was immunoprecipitated under native conditions, analysed by SDS–PAGE, and quantified by phosphorimaging. The ATPase activity of Ni-NTA-purified protein was determined using an NADH-coupled microplate photometric assay36 (link),37 (link)Wild-type and mutant GET3 genes were subcloned into a low copy number URA plasmid under the control of a medium-strength, constitutive ACT1 promoter, and transformed into a Δget3 strain (Open Biosystems); serial dilutions of each transformant were spotted (along with wild type and vector only controls) onto synthetic defined medium (−uracil) supplemented with 100 μg ml−1 hygromycin B. Plates were photographed after 2 days at 37 °C.
Publication 2009
adenosine 5'-O-(2-thiodiphosphate) Adenosinetriphosphatase Biological Assay Chromatography Cloning Vectors Diffusion Escherichia coli Gel Chromatography Genes Genes, vif GTP-Binding Protein alpha Subunit, Gi Helix (Snails) Homo sapiens Hygromycin B Lanugo Mutagenesis Mutant Proteins Mutation NADH Nucleotides Photometry Plasmids Proteins Reticulocytes Schizosaccharomyces pombe SDS-PAGE Selenomethionine Strains Technique, Dilution Uracil
Sexual differentiation was induced in tightly synchronized P. falciparum parasites (28±2 hpi) by incubating the cells for 22 hours in CM (220 μL per well of a 96-well plate; 0.3-0.5% parasitemia; 2.5% hematocrit) or in −SerM as described (Brancucci et al., 2015 (link)). If not stated otherwise, cell line Pf2004/164tdTom was used for all experiments. To determine the effect of culture perturbations on sexual commitment, serum fractions as well as nutrients or inhibitor compounds (solved in either RPMI, DMSO, chloroform, ethanol or methanol) were added to the bottom of empty wells (glass bottom dishes were used for chloroform-containing samples) and directly resuspended in parasite culture after allowing volatile solvents to evaporate. To determine sexual differentiation in reticulocyte-enriched blood, tightly synchronized parasites were magnet purified at 46±2 hpi using MACS CS columns in a SuperMACS (Miltenyi Biotec) before incubating pure schizont-infected erythrocytes (>99%) with the blood sample to be tested. These culture perturbations were then tested for effects on parasite sexual differentiation as described (Brancucci et al., 2015 (link)). In brief, following the 22 hour testing phase (see above), cells of each well were washed 3 times in 200 μL +SerM medium before being resuspended in 220 μL +SerM medium. Henceforth, medium was exchanged daily. Parasitemia and gametocytemia was quantified using flow cytometry at 20-30 hpi (MACS Quant, Analyzer 10) and 72-96 hpi (BD Fortessa), respectively. Cytometry data were analysed using FlowJo software and sexual differentiation rates were determined by dividing gametocytemia of each well with the corresponding parasitemia measurements. Assays were run in biological triplicates. Each biological replicate contained technical triplicates.
P. berghei sexual commitment assays were performed using a parasite line expressing an RFP reporter under the gametocyte-specific gene PBANKA_1018700 (Sinha et al., 2014 (link)) and GFP under the constitutive PBANKA_0905600 promoter, in the 507cl1 background line (RMgm-7). Mature schizonts were intravenously (IV) administered to naïve TO mice. Ring stage parasites were isolated at 4 hpi and mature trophozoites and gametocytes were removed by passing through a MACS LD column (Miltenyi Biotec). Infected erythrocytes were incubated in −SerM medium, −SerM medium supplemented with 20 μM LysoPC (−SerM/LysoPC), or serum-complemented medium (+SerM) for 20 hours. Mature schizont stage parasites were then isolated on a 55% Nycodenz (Axis-Shield POC)/RPMI gradient and injected intravenously into 2 or 3 naïve mice. GFP-expressing cells were examined by flow cytometry at 16 hpi to calculate parasitemia, while cells expressing both RFP and GFP (gametocytes) were assessed at 21 hpi. Gametocytemia was calculated as [(RFP+ and GFP+ cells)/GFP+ cells]100.
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Publication 2017
Biological Assay Biopharmaceuticals BLOOD Cell Lines Cells Chloroform DNA Replication Epistropheus Erythrocytes Ethanol Flow Cytometry Genes, Reporter Hyperostosis, Diffuse Idiopathic Skeletal LY353381hydrochloride LYSO-PC Methanol Mus Nutrients Nycodenz Parasitemia Parasites Reticulocytes Schizonts Selective Estrogen Receptor Modulators Serum Sex Differentiation Solvents Sulfoxide, Dimethyl Trophozoite Volumes, Packed Erythrocyte

Most recents protocols related to «Reticulocytes»

mRNA was extracted from neutrophils and reticulocytes and transcribed with superScript III first‐strand synthesis SuperMix, according to the manufacturer's instructions (Thermo Fisher Scientific). cDNA products were amplified by PCR using a primer combination specific for the P1 and P2 SLC44A2 transcripts. Primer sequences are available in Appendix Table S2.
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Publication 2023
Anabolism DNA, Complementary Neutrophil Oligonucleotide Primers Reticulocytes RNA, Messenger
Human neutrophils were isolated from fresh whole blood (< 4 h after blood sampling) using the MACSxpress Neutrophil Isolation Kit followed by a MACSxpress Erythrocyte Depletion Kit (Miltenyi Biotec). Reticulocytes were isolated from whole blood using the CD71 MicroBeads Kit (Miltenyi Biotec). The purity of the cells, determined by flow cytometry using anti‐CD16 and anti‐CD71 antibodies, was approximately 98%.
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Publication 2023
Anti-Antibodies Blood Erythrocytes Flow Cytometry Homo sapiens isolation Microspheres Neutrophil Reticulocytes TFRC protein, human
GST fusion proteins were expressed in BL21 (Vazyme) Escherichia coli, followed by ultrasonic disruption and purification using glutathione Sepharose 4B beads (GE). In vitro transcription and translation experiments were performed using rabbit reticulocyte lysate (TNT systems, Promega) according to the manufacturer’s recommendation. The beads were co-incubated with the in vitro translated proteins and then washed with binding buffer. The eluates were then analyzed using SDS-PAGE.
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Publication 2023
Buffers Escherichia coli Glutathione Promega Proteins Rabbits Reticulocytes SDS-PAGE Sepharose 4B Transcription, Genetic Ultrasonics
Recombinant GST-N-Myc protein was incubated with or without recombinant p300 protein in a ubiquitination reaction containing 20 mM Hepes (pH 7.5, catalog no.: H3375; Sigma–Aldrich), 5 mM MgCl2 (catalog no.: AM9530G; Thermo Fisher Scientific), 2 mM dithiothreitol (catalog no.: R0861; Thermo Fisher Scientific), 2 mM ATP (catalog no.: A6559; Sigma–Aldrich), 5 mg of ubiquitin (catalog no.: U5507; Sigma–Aldrich), 20 mM MG132, and 5 μl/reaction crude rabbit reticulocyte lysate (catalog no.: L4151; Promega) for 1 h at 30 °C (24 (link)). Products were then subjected to IP using GST antibody (catalog no.: 2624; Cell Signaling Technology) and protein A/G magnetic beads before Western blot.
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Publication 2023
Dithiothreitol EP300 protein, human G-substrate GTP-Binding Proteins HEPES Immunoglobulins Magnesium Chloride MG 132 Promega Rabbits Recombinant Proteins Reticulocytes Staphylococcal Protein A Ubiquitin Ubiquitination Western Blotting
Capped, poly(A)-tailed Homo sapiens CREB3L2 (HA- or V5-tagged, cleaved form) and ATF4 (HA- or V5-tagged; engineered from Addgene plasmid #26114, gifted by Y. Ye) transcripts were synthesized using the mMESSAGE mMACHINE SP6 and Poly(A) Tailing kits from Thermo Fisher Scientific. RNAs were column-purified in accordance to the manufacturer’s instructions (RNeasy Mini Kit, QIAGEN), and efficient poly(A) tailing was evaluated by agarose gel electrophoresis. CREB3L2 and ATF4 translation reactions were incubated separately for 90 min at 30°C following vendor’s guidelines (Rabbit Reticulocyte Lysate System, Promega). CREB3L2 and ATF4 protein products were confirmed by immunoblotting in pilot experiments. Upon completion of the translation protocol, lysates (45 μl) containing CREB3L2 and ATF4 were mixed and incubated at 37°C for 30 min with gentle agitation (300 rpm for 5 s every minute) before immunoprecipitation, as previously described (71 (link)). Immunoprecipitation was carried out overnight at 4°C with gentle rotation in PBS supplemented with 0.1% NP-40 and protease inhibitors (cOmplete, EDTA-free Protease Inhibitor Cocktail, Roche), using magnetic beads conjugated with anti-HA or anti-V5 antibodies (anti-HA beads: PI88836, Thermo Fisher Scientific; anti-V5 beads: NC0777490, MBL International). Washing cycles were repeated five times with immunoprecipitation buffer. Complex elution was performed in 2× Laemmli buffer [130 mM tris-Cl (pH 6.8), 0.1 mM dithiothreitol, 20% (v/v) glycerol, and 4% SDS diluted in water] by boiling at 95°C for 5 min. Immunoblot detection of tagged CREB3L2 and ATF4: anti-HA (1:4000; ab9110, Abcam), anti-V5 (R960-25, Thermo Fisher Scientific), and anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:10,000; 60004-1-Ig, Proteintech), in conjugation with TrueBlot anti-rabbit IgG horseradish peroxidase (HRP) (1:1000; Rockland) or Superclonal anti-mouse IgG HRP (1:10,000; A28177, Thermo Fisher Scientific).
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Publication 2023
Anti-Antibodies ATF4 protein, human Buffers Dithiothreitol Edetic Acid Electrophoresis, Agar Gel Glyceraldehyde-3-Phosphate Dehydrogenases Glycerin Homo sapiens IGG-horseradish peroxidase Immunoblotting Immunoprecipitation Laemmli buffer Mus Nonidet P-40 Plasmids Promega Protease Inhibitors Proteins Rabbits Reticulocytes RNA Tromethamine

Top products related to «Reticulocytes»

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The TNT Coupled Reticulocyte Lysate System is a cell-free protein expression system that enables the in vitro synthesis of proteins from DNA templates. It uses a rabbit reticulocyte lysate to provide the necessary cellular components for transcription and translation of the target protein.
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Rabbit reticulocyte lysate is a cell-free protein synthesis system derived from rabbit reticulocytes, which are immature red blood cells. The lysate contains the necessary components for in vitro translation of mRNA, enabling the synthesis of proteins from exogenously added templates.
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The TNT T7 Coupled Reticulocyte Lysate System is a lab equipment product that enables the in vitro expression of proteins from DNA templates. It provides the necessary components for coupled transcription and translation of target genes under the control of the T7 RNA polymerase promoter.
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The Rabbit Reticulocyte Lysate System is a cell-free protein synthesis system. It enables the in vitro translation of mRNA into proteins using a lysate derived from rabbit reticulocytes.
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The Flexi Rabbit Reticulocyte Lysate System is a cell-free protein expression system derived from rabbit reticulocytes. It is designed to enable efficient in vitro translation of mRNA into functional proteins.
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[35S]-methionine is a radioactive isotope of the amino acid methionine. It is commonly used as a labeling agent in various biochemical and molecular biology applications.
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The TNT Systems are a range of laboratory equipment designed for the sensitive and specific detection of various targets, including proteins, nucleic acids, and other biomolecules. These systems employ the principles of chemiluminescence or fluorescence to provide a quantitative measure of the target analytes. The core function of the TNT Systems is to facilitate accurate and reliable analysis of samples in a variety of research and diagnostic applications.
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The Luciferase Assay System is a laboratory tool designed to measure the activity of the luciferase enzyme. Luciferase is an enzyme that catalyzes a bioluminescent reaction, producing light. The Luciferase Assay System provides the necessary reagents to quantify the level of luciferase activity in samples, enabling researchers to study biological processes and gene expression.
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The TNT Quick Coupled Transcription/Translation System is a laboratory tool that enables the simultaneous in vitro transcription and translation of proteins from DNA templates. It provides a rapid and efficient means of producing proteins for various research applications.
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The Retic-COUNT is a laboratory equipment designed to automate the counting of reticulocytes, a type of immature red blood cells. It utilizes flow cytometry technology to rapidly and accurately analyze blood samples, providing quantitative data on reticulocyte count and related parameters.

More about "Reticulocytes"

Reticulocytes are immature red blood cells that serve as an important indicator of erythropoiesis, the process of red blood cell production.
These transitional cells contain remnants of the nucleus and ribosomal RNA, giving them a reticulated or reticular appearance under a microscope.
Reticulocyte count is a crucial diagnostic marker used by clinicians and researchers to gain insights into various hematological conditions, such as anemia, hemolysis (the premature destruction of red blood cells), and blood loss.
By studying reticulocyte dynamics, researchers can optimize their research protocols and enhance the reproducibility of their work.
The TNT Coupled Reticulocyte Lysate System and the Rabbit Reticulocyte Lysate System are commonly used in vitro translation systems that harness the protein synthesis capabilities of reticulocytes.
These systems, along with the TNT T7 Coupled Reticulocyte Lysate System, Flexi Rabbit Reticulocyte Lysate System, and the Luciferase Assay System, provide researchers with versatile tools to study gene expression, protein synthesis, and other cellular processes.
The incorporation of [35S]-methionine, a radioactive amino acid, into the proteins synthesized by these reticulocyte-based systems allows for the detection and quantification of newly translated proteins.
The TNT Quick Coupled Transcription/Translation System and the Retic-COUNT assay further expand the capabilities of reticulocyte-based research, enabling efficient and streamlined analysis of gene expression and cell viability.
By leveraging the insights gained from the understanding of reticulocytes and the available research tools, scientists can optimize their protocols, enhance reproducibility, and gain valuable insights into hematological conditions and cellular processes.