Phenomenex

Torrance, CA, United States

Phenomenex

Torrance, CA, United States

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News Article | May 9, 2017
Site: www.chromatographytechniques.com

Phenomenex’s new food testing guide helps the industry meet increasing demands for quality and safety testing and adhere to the guidelines of the Food Safety Modernization Act (FMSA). The 160-page guide presents more than 150 applications using HPLC, LC/MS, UHPLC/MS, GC, GC/MS and sample preparation techniques and covers a wide range of compound classes including contaminants, mycotoxins, pesticides, veterinary pharmaceuticals, sugars, dietary supplements and vitamins, along with new GC tools for fast fatty acids analysis. Phenomenex, Inc. www.phenomenex.com, 310-212-0555


News Article | May 9, 2017
Site: www.chromatographytechniques.com

Phenomenex’s new food testing guide helps the industry meet increasing demands for quality and safety testing and adhere to the guidelines of the Food Safety Modernization Act (FMSA). The 160-page guide presents more than 150 applications using HPLC, LC/MS, UHPLC/MS, GC, GC/MS and sample preparation techniques and covers a wide range of compound classes including contaminants, mycotoxins, pesticides, veterinary pharmaceuticals, sugars, dietary supplements and vitamins, along with new GC tools for fast fatty acids analysis. Phenomenex, Inc. www.phenomenex.com, 310-212-0555


News Article | May 10, 2017
Site: www.marketwired.com

TORRANCE, CA--(Marketwired - May 09, 2017) - Phenomenex Inc., global leader in the research, design and manufacture of advanced technologies for the separation sciences, introduces new mixed-mode selectivities and particle sizes for UHPLC, HPLC and preparative work in the Luna column family. The first is the new Luna Omega Polar C18 stationary phase -- a unique selectivity on an innovative silica particle that delivers a wide elution window and combined high retention for polar and nonpolar analytes. This new phase is 100 percent aqueous-stable due to a polar-modified surface, providing flexibility in solvent and gradient system selection needed to achieve desired polar/non polar analyte separation. The Luna Omega Polar C18 is offered in a high-performance 1.6µm particle for UHPLC instruments as well as a low-pressure 5µm particle for direct scalability to analytical HPLC or preparative work. The 5µm particle is available in Phenomenex's patented Axia-packed preparative columns. Phenomenex is also introducing the 100 percent aqueous-stable Luna Omega PS C18 that delivers two distinct separation mechanisms at the same time. The particle surface of the PS C18 contains a positive charge that facilitates greater acidic compound retention through ionic interaction, while the C18 ligand delivers general reversed-phase retention. This mixed-mode selectivity is a valuable tool for greater separation between mixtures of compounds that have varying functional groups, such as peptides, pesticides or metabolite profiles. Additionally, the positive surface charge encourages excellent basic compound peak shape through the ionic repulsion of these compound species. Luna Omega columns are well suited for a wide range of applications including drug discovery and development, food contaminant analysis, environmental testing, toxicology and clinical research. "Our Luna HPLC brand is one of the most recognized in the global chromatography industry, with a 20-year track record," commented Simon Lomas, strategic marketing manager for Phenomenex. "The recent expansion of this column family with novel Luna Omega silica particle technology and mixed-mode polar stationary phases has enabled us to bring the Luna legacy to the UHPLC world." Phenomenex Kinetex® Core-Shell Technology and Luna Omega columns together provide an ideal complementary UHPLC solution for greater efficiency and separation power. With a growing range of valuable selectivities, this pairing of core-shell and fully porous UHPLC products affords customers a better choice for immediate gains in productivity, resolution and retention. Combining the Luna Omega Polar C18 with the previously released fully porous Luna Omega 1.6µm C18 and Kinetex 1.7µm core-shell phases further expands the options for UHPLC method development and improvement. Phenomenex is a global technology leader committed to developing novel analytical chemistry solutions that solve the separation and purification challenges of researchers in industrial, clinical research, government and academic laboratories. From drug discovery and pharmaceutical development to food safety and environmental analysis, Phenomenex chromatography solutions accelerate science and help researchers improve global health and well-being. For more information on Phenomenex, visit www.phenomenex.com or follow the company on Twitter @Phenomenex.


News Article | May 12, 2017
Site: www.marketwired.com

TORRANCE, CA--(Marketwired - May 12, 2017) - Phenomenex Inc., a global leader in the research and manufacture of advanced technologies for the separation sciences, introduces the Kinetex 2.6 µm Polar C18 -- the ninth selectivity in the Kinetex core-shell family. This stationary phase combines C18 ligands with a polar-modified surface to enable superior retention of polar and nonpolar compounds while ensuring 100 percent aqueous stability. Incorporating the key performance benefits of the Kinetex Core-Shell Technology, the 2.6 µm Polar C18 particle size provides high efficiency and performance on HPLC systems and potential increases in resolution, sensitivity and separation speed. On UHPLC systems, the 2.6 µm size provides comparable performance to fully porous sub-2 µm particles, but at much lower backpressure levels. With this new Kinetex particle, Phenomenex now offers the dual polar/nonpolar selectivity of the Polar C18 stationary phase in two particle types -- Kinetex core-shell and Luna Omega thermally modified fully porous. This enables researchers to move back and forth between two different solid supports to better address method development needs and wants. "We are pleased to extend the performance and sensitivity of our Kinetex Core-Shell Technology products to the challenges of retaining and analyzing polar compounds," comments Simon Lomas, strategic marketing manager for Phenomenex. "This new selectivity greatly enhances polar/nonpolar retention compared to traditional alkyl phases and gives customers a versatile product with which they can start reversed phase method development." The new Kinetex Polar C18 is an ideal all-purpose phase for use with mixtures of multiple polar and nonpolar compounds or single-class methods with closely related compounds, such as impurities or metabolites. Key applications that can greatly benefit from this dual selectivity include pesticide screening in food, testing of emerging environmental contaminants and drug analysis in pharmaceutical discovery, toxicology testing and clinical research. For more information, visit www.phenomenex.com/kinetex. Phenomenex is a global technology leader committed to developing novel analytical chemistry solutions that solve the separation and purification challenges of researchers in industrial, clinical research, government and academic laboratories. From drug discovery and pharmaceutical development to food safety and environmental analysis, Phenomenex chromatography solutions accelerate science and help researchers improve global health and well-being. For more information on Phenomenex, visit www.phenomenex.com or follow the company on Twitter @Phenomenex.


TORRANCE, CA--(Marketwired - May 11, 2017) - Phenomenex Inc., global leader in the research, design and manufacture of advanced technologies for the separation sciences, introduces two new particle chemistries for the characterization and purification of synthetic oligonucleotides. Joining the company's Clarity BioSolutions portfolio are Clarity Oligo-XT, for high-efficiency reversed-phase LC analysis and purification, and Clarity Oligo-SAX, a high-resolution strong anion exchanger (SAX) for characterization. Phenomenex Clarity BioSolutions, first introduced almost a decade ago, are used in pharmaceutical labs and contract research organizations (CROs) where synthetic oligonucleotide-based therapeutics are being developed and tested, as well as in the labs of custom oligonucleotide and nucleic acid manufacturers. Synthetic oligonucleotides are growing in popularity as novel agents for the treatment of disease, and liquid chromatography continues to be an effective tool for characterization, with technology advancements that address improvements in resolution, throughput and column lifetime. The new Clarity Oligo-XT C18 columns feature novel and robust core-shell media, for high-efficiency reversed-phase characterization of synthetic DNA and RNA, pH stability from 1 to 12 and increased sensitivity that improves quantitation by mass spectrometry (MS). These core-shell particles deliver the separation power necessary to accurately resolve closely related synthetic oligonucleotide sequences. Clarity Oligo-XT columns are available in directly scalable 1.7µm, 2.6µm and 5µm particle sizes that enable easy method transfer between analytical HPLC/UHPLC instrumentation and preparative purifications systems. The 5µm particle is available in Phenomenex's patented Axia-packed preparative columns that increase purification performance. The Clarity Oligo-SAX columns feature an entirely new, rugged non-porous particle that retains synthetic oligonucleotides through strong ion exchange mechanisms, adding a robust strong anion exchanger choice with improved column lifetimes to the Clarity family. These quaternary amine functionalized, nonporous particles are engineered for performance at high pH (2.5 to 12.5) and temperatures up to 85 ˚C and are provided in 5µm particle size for analytical characterization. "Our synthetic oligonucleotide characterization products are solution-based and have been developed to meet our customers' evolving needs," said Simon Lomas, strategic marketing manager for Phenomenex. "The Oligo-XT and Oligo-SAX columns allow customers to capitalize on both reversed-phase and ion exchange chromatography to get greater separation, faster analyses and extended column lifetimes." Phenomenex's Clarity BioSolutions product portfolio delivers purified synthetic oligonucleotides in a range of formats and scales to meet the specific demands of the industry. From simple desalting, to rapid isolation of oligonucleotide therapeutics from biological samples, to high-throughput, high-purity purification techniques, the Clarity BioSolutions portfolio offers a comprehensive approach. Phenomenex is a global technology leader committed to developing novel analytical chemistry solutions that solve the separation and purification challenges of researchers in industrial, clinical research, government and academic laboratories. From drug discovery and pharmaceutical development to food safety and environmental analysis, Phenomenex chromatography solutions accelerate science and help researchers improve global health and well-being. For more information on Phenomenex, visit www.phenomenex.com or follow the company on Twitter @Phenomenex.


TORRANCE, CA--(Marketwired - May 18, 2017) - Phenomenex Inc., global leader in the research, design and manufacture of advanced technologies for the separation sciences, is introducing two new offerings to its Luna Omega fully porous LC column line -- the Luna Omega 3 µm Polar C18 and the Luna Omega 3 µm PS C18. The Luna Omega Polar C18 stationary phase is a unique, robust selectivity bonded to an innovative silica particle that delivers high loadability and retention for both polar and nonpolar analytes. This new phase is 100 percent aqueous-stable due to a polar-modified surface, providing flexibility in solvent and gradient system selection needed to achieve desired polar/nonpolar analyte separation. The new 3 µm particle joins the existing 1.6 µm and 5 µm sizes to provide full scalability from UHPLC to HPLC to preparative chromatography. Unlike a traditional C18, the Luna Omega PS C18 delivers two distinct and useful separation mechanisms and offers 100 percent aqueous stability. The particle surface of the PS C18 contains a positive charge that facilitates greater acidic compound retention through ionic interaction, while the C18 ligand delivers general reversed-phase retention. This mixed-mode selectivity is a valuable tool for greater separation between mixtures of compounds that have varying functional groups, such as peptides, pesticides or metabolite profiles. Additionally, the positive surface charge encourages sharp basic compound peak shape through the ionic repulsion of these compound species. Like the Luna Omega Polar C18, the three PS 18 particle size choices enable full scalability from UHPLC to HPLC to preparative chromatography. Luna Omega columns are well suited for a wide range of applications including drug discovery and development, food contaminant analysis, environmental testing, toxicology and clinical research. "With three directly scalable particle sizes, chromatographers can easily take advantage of the enhanced retention of the Polar C18 and PS C18 for their HPLC and UHPLC work," commented Simon Lomas, strategic marketing manager for Phenomenex. "These new selectivities offer our customers high-efficiency solutions for their large range of work, from routine/general purpose to complex analyses." For more information, visit www.phenomenex.com/lunaomega. Phenomenex is a global technology leader committed to developing novel analytical chemistry solutions that solve the separation and purification challenges of researchers in industrial, clinical research, government and academic laboratories. From drug discovery and pharmaceutical development to food safety and environmental analysis, Phenomenex chromatography solutions accelerate science and help researchers improve global health and wellbeing. For more information please visit www.phenomenex.com and follow the company's blog at www.scienceunfiltered.com Link to this release at: https://www.phenomenex.com/Home/News?id=New_3_µm_Particle_Size_Adds_HPLC_Scalability_to_Luna_Omega_Mixed-Mode_Selectivities Please send READER INQUIRIES from this news release directly to: info@phenomenex.com


News Article | May 27, 2017
Site: www.marketwired.com

TORRANCE, CA--(Marketwired - May 27, 2017) - Phenomenex Inc., a global leader in the research and manufacture of advanced technologies for the separation sciences, announces the opening of a new manufacturing and development facility dedicated to the company's gas chromatography (GC) columns, marketed under the Zebron™ brand name. The 15,000-square-foot facility, which was designed and extensively renovated specifically for Phenomenex, is located in the Sacramento, Calif. suburb of El Dorado Hills. Prior to the expansion, GC products were manufactured in Sutter Creek, Calif. The new location supports twice the production capacity and enables improved logistics and delivery speeds to the company's global customers. Phenomenex is currently expanding sales internationally as demand for GC columns grows in North America and Europe as well as in India and China, particularly in food testing applications. "The Phenomenex GC manufacturing and development operation has a rich and long history," comments Emmet Welch, senior product development manager for Phenomenex. "We are staffed with scientists and production experts with decades of leadership in every aspect of GC column technology and manufacturing. In fact, many of our current staff have more than 25 years experience in column technology development." The new facility includes organic synthesis, R&D and analytical labs. "Detailed and exhaustive planning went into creating an efficient production floor, using lean principles to maximize the use of space while minimizing the movement of people and materials. We have also included a centralized piping system that reduces the cost and movement of process gasses," continued Welch. "With advanced, automated workflows, this new facility will be capable of supporting significant growth in Phenomenex GC manufacturing and new product development for many years." The facility's work environment for employees features a bright, colorful and open design, complete with inspiring, art-filled spaces and an outside athletic center. Phenomenex founder, Fasha Mahjoor, remarked, "Phenomenex is known for the vibrant colors and pleasing architectural spaces that are the hallmarks of our corporate headquarters. Our people are the reason for our success, and it's our goal and responsibility to give them an environment that inspires teamwork and camaraderie and promotes their health and well-being." About Phenomenex Phenomenex is a global technology leader committed to developing novel analytical chemistry solutions that solve the separation and purification challenges of researchers in industrial, clinical research, government and academic laboratories. From drug discovery and pharmaceutical development to food safety and environmental analysis, Phenomenex chromatography solutions accelerate science and help researchers improve global health and wellbeing. For more information please visit www.phenomenex.com and follow the company's blog at www.scienceunfiltered.com


News Article | February 22, 2017
Site: www.nature.com

Pre-B acute lymphoblastic leukaemia (ALL) cells were obtained from patients who gave informed consent in compliance with the guidelines of the Internal Review Board of the University of California San Francisco (Supplementary Table 2). Leukaemia cells from bone marrow biopsy of patients with ALL were xenografted into sublethally irradiated NOD/SCID (non-obese diabetic/severe combined immunodeficient) mice via tail vein injection. After passaging, leukaemia cells were collected. Cells were cultured on OP9 stroma cells in minimum essential medium-α (MEMα; Invitrogen), supplemented with 20% fetal bovine serum (FBS), 2 mM l-glutamine, 1 mM sodium pyruvate, 100 IU/ml penicillin and 100 μg/ml streptomycin. Primary chronic myeloid leukaemia (CML) cases were obtained with informed consent from the University Hospital Jena in compliance with institutional internal review boards (including the IRB of the University of California San Francisco; Supplementary Table 3). Cells were cultured in Iscove’s modified Dulbecco’s medium (IMDM; Invitrogen) supplemented with 20% BIT serum substitute (StemCell Technologies); 100 IU/ml penicillin and 100 μg/ml streptomycin; 25 μmol/l β-mercaptoethanol; 100 ng/ml SCF; 100 ng/ml G-CSF; 20 ng/ml FLT3; 20 ng/ml IL-3; and 20 ng/ml IL-6. Human cell lines (Supplementary Table 2) were obtained from DSMZ and were cultured in Roswell Park Memorial Institute medium (RPMI-1640; Invitrogen) supplemented with GlutaMAX containing 20% FBS, 100 IU/ml penicillin and 100 μg/ml streptomycin. Cell cultures were kept at 37 °C in a humidified incubator in a 5% CO atmosphere. None of the cell lines used was found in the database of commonly misidentified cell lines maintained by ICLAC and NCBI Biosample. All cell lines were authenticated by STR profiles and tested negative for mycoplasma. BML275 (water-soluble) and imatinib were obtained from Santa Cruz Biotechnology and LC Laboratories, respectively. Stock solutions were prepared in DMSO or sterile water at 10 mmol/l and stored at −20 °C. Prednisolone and dexamethasone (water-soluble) were purchased from Sigma-Aldrich and were resuspended in ethanol or sterile water, respectively, at 10 mmol/l. Stock solutions were stored at −20 °C. Fresh solutions (pH-adjusted) of methyl pyruvate, OAA, 3-OMG (an agonist of TXNIP), d-allose (an agonist of TXNIP) and recombinant insulin (Sigma-Aldrich) were prepared for each experiment. DMS was obtained from Acros Organics, and fresh solutions (pH-adjusted) were prepared before each experiment. For competitive-growth assays, 5 mmol/l methyl pyruvate, 5 mmol/l dimethyl succinate (DMS) and 5 mmol/l OAA were used. The CNR2 agonist HU308 was obtained from Cayman Chemical. To avoid inflammation-related effects in mice, bone marrow cells were extracted from mice (Supplementary Table 4) younger than 6 weeks of age without signs of inflammation. All mouse experiments were conducted in compliance with institutional approval by the University of California, San Francisco Institutional Animal Care and Use Committee. Bone marrow cells were obtained by flushing cavities of femur and tibia with PBS. After filtration through a 70-μm filter and depletion of erythrocytes using a lysis buffer (BD PharmLyse, BD Biosciences), washed cells were either frozen for storage or subjected to further experiments. Bone marrow cells were cultured in IMDM (Invitrogen) with GlutaMAX containing 20% fetal bovine serum, 100 IU/ml penicillin, 100 μg/ml streptomycin and 50 μM β-mercaptoethanol. To generate pre-B ALL (Ph+ ALL-like) cells, bone marrow cells were cultured in 10 ng/ml recombinant mouse IL-7 (PeproTech) and retrovirally transformed by BCR–ABL1. BCR–ABL1-transformed pre-B ALL cells were propagated only for short periods of time and usually not for longer than 2 months to avoid acquisition of additional genetic lesions during long-term cell culture. To generate myeloid leukaemia (CML-like) cells, the myeloid-restricted protocol described previously30 was used. Bone marrow cells were cultured in 10 ng/ml recombinant mouse IL-3, 25 ng/ml recombinant mouse IL-6, and 50 ng/ml recombinant mouse SCF (PeproTech) and retrovirally transformed by BCR–ABL1. Immunophenotypic characterization was performed by flow cytometry. For conditional deletion, a 4-OHT-inducible, Cre-mediated deletion system was used. For retroviral constructs used, see Supplementary Table 5. Transfection of retroviral constructs (Supplementary Table 5) was performed using Lipofectamine 2000 (Invitrogen) with Opti-MEM medium (Invitrogen). Retroviral supernatant was produced by co-transfecting HEK 293FT cells with the plasmids pHIT60 (gag-pol) and pHIT123 (ecotropic env). Lentiviral supernatant was produced by co-transfecting HEK 293FT cells with the plasmids pCDNL-BH and VSV-G or EM140. 293FT cells were cultured in high glucose Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) with GlutaMAX containing 10% fetal bovine serum, 100 IU/ml penicillin, 100 μg/ml streptomycin, 25 mmol/l HEPES, 1 mmol/l sodium pyruvate and 0.1 mmol/l non-essential amino acids. Regular medium was replaced after 16 h by growth medium containing 10 mmol /l sodium butyrate. After incubation for 8 h, the medium was changed back to regular growth medium. After 24 h, retroviral supernatant was collected, filtered through a 0.45-μm filter and loaded by centrifugation (2,000g, 90 min at 32 °C) onto 50 μg/ml RetroNectin- (Takara) coated non-tissue 6-well plates. Lentiviral supernatant produced with VSV-G was concentrated using Lenti-X Concentrator (Clontech), loaded onto RetroNectin-coated plates and incubated for 15 min at room temperature. Lentiviral supernatant produced with EM140 was collected, loaded onto RetroNectin-coated plates and incubated for 30 min at room temperature. Per well, 2–3 × 106 cells were transduced by centrifugation at 600g for 30 min and maintained for 48 h at 37 °C with 5% CO before transferring into culture flasks. For cells transduced with lentiviral supernatant produced with EM140, supernatant was removed the day after transduction and replaced with fresh culture medium. Cells transduced with oestrogen-receptor fusion proteins were induced with 4-OHT (1 μmol/l). Cells transduced with constructs carrying an antibiotic-resistance marker were selected with its respective antibiotic. For loss-of-function studies, dominant-negative variants of IKZF1 (DN-IKZF1, lacking the IKZF1 zinc fingers 1–4) and PAX5 (DN-PAX5; PAX5–ETV6 fusion) were cloned from patient samples. Expression of DN-IKZF1 was induced by doxycycline (1 μg/ml), while activation of DN-PAX5 was induced by 4-OHT (1 μg/ml) in patient-derived pre-B ALL cells carrying IKZF1 and PAX5 wild-type alleles, respectively. Inducible reconstitution of wild-type IKZF1 and PAX5 in haploinsufficient pre-B ALL cells carrying deletions of either IKZF1 (IKZF1∆) or PAX5 (PAX5∆) were also studied. Lentiviral constructs used are listed in Supplementary Table 5. A doxycycline-inducible TetOn vector system was used for inducible expression of PAX5 in mouse BCR–ABL1 pre-B ALL. The retroviral constructs used are listed in Supplementary Table 5. To study the effects of B-cell- versus myeloid-lineage identity in genetically identical mouse leukaemia cells, a doxycycline-inducible TetOn-CEBPα vector system31 was used to reprogram B cells. Mouse BCR–ABL1 pre-B ALL cells expressing doxycycline-inducible CEBPα or an empty vector were induced with doxycycline (1 μg/ml). Conversion from the B-cell lineage (CD19+Mac1−) to the myeloid lineage (CD19−Mac1+) was monitored by flow cytometry. For western blots, B-lineage cells (CD19+Mac1−) and CEBPα-reprogrammed cells (CD19−Mac1+) were sorted from cells expressing an empty vector or CEBPα, respectively, following doxycycline induction. For metabolic assays, sorted B-lineage cells and CEBPα-reprogrammed cells were cultured (with doxycycline) for 2 days following sorting, and were then seeded in fresh medium for measurement of glucose consumption (normalized to cell numbers) and total ATP levels (normalized to total protein). To study Lkb1 deletion in the context of CEBPα-mediated reprogramming, BCR–ABL1-transformed Lkb1fl/fl pre-B ALL cells expressing doxycycline-inducible CEBPα were transduced with 4-OHT(1 μg/ml) inducible Cre-GFP (Cre-ERT2-GFP). Without sorting for GFP+ cells, cells were induced with doxycycline and 4-OHT. Viability (expressed as relative change of GFP+ cells) was measured separately in B-lineage (gated on CD19+ Mac1−) and myeloid lineage (gated on CD19− Mac1+) populations. To study whether Lkb1 deletion causes CEBPα-dependent effects on metabolism and signalling, Lkb1fl/fl BCR–ABL1 B-lineage ALL cells expressing doxycycline-inducible CEBPα or an empty vector were transduced with 4-OHT-inducible Cre-GFP. After sorting for GFP+ populations, cells were induced with doxycycline. B-lineage cells (CD19+ Mac1−) and CEBPα-reprogrammed cells (CD19− Mac1+) were sorted from cells expressing an empty vector or CEBPα, respectively. Sorted cells were cultured with doxycycline and induced with 4-OHT. Protein lysates were collected on day 2 following 4-OHT induction. For metabolomics, sorted cells were re-seeded in fresh medium on day 2 following 4-OHT induction and collected for metabolite extraction. For CRISPR/Cas9-mediated deletion of target genes, all constructs including lentiviral vectors expressing gRNA and Cas9 nuclease were purchased from Transomic Technologies (Supplementary Table 5; see Supplementary Table 6 for gRNA sequences). In brief, patient-derived pre-B ALL cells transduced with GFP-tagged, 4-OHT-inducible PAX5 or an empty vector were transduced with pCLIP-hCMV-Cas9-Nuclease-Blast. Blasticidin-resistant cells were subsequently transduced with pCLIP-hCMV-gRNA-RFP. Non-targeting gRNA was used as control. Constructs including lentiviral vectors expressing gRNA and dCas9-VPR used for CRISPR/dCas9-mediated activation of gene expression are listed in Supplementary Table 5. Nuclease-null Cas9 (dCas9) fused with VP64-p65-Rta (VPR) was cloned from SP-dCas9-VPR (a gift from G. Church; Addgene plasmid #63798) and then subcloned into pCL6 vector with a blasticidin-resistant marker. gRNA sequences (Supplementary Table 6) targeting the transcriptional start site of each specific gene were obtained from public databases (http://sam.genome-engineering.org/ and http://www.genscript.com/gRNA-database.html)32. gBlocks Gene Fragments were used to generate single-guide RNAs (sgRNAs) and were purchased from Integrated DNA Technologies, Inc. Each gRNA was subcloned into pCL6 vector with a dsRed reporter. Patient-derived pre-B ALL cells transduced with either GFP-tagged inducible PAX5 or an empty vector were transduced with pCL6-hCMV-dCas9-VPR-Blast. Blasticidin-resistant cells were used for subsequent transduction with pCL6-hCMV-gRNA-dsRed, and dsRed+ cells were further analysed by flow cytometry. For each target gene, 2–3 sgRNA clones were pooled together to generate lentiviruses. Non-targeting gRNA was used as control. To elucidate the mechanistic contribution of PAX5 targets, the percentage of GFP+ cells carrying gRNA(s) for each target gene was monitored by flow cytometry upon inducible activation of GFP-tagged PAX5 or an empty vector in patient-derived pre-B ALL cells in competitive-growth assays. Cells were lysed in CelLytic buffer (Sigma-Aldrich) supplemented with a 1% protease inhibitor cocktail (Thermo Fisher Scientific). A total of 20 μg of protein mixture per sample was separated on NuPAGE (Invitrogen) 4–12% Bis-Tris gradient gels or 4–20% Mini-PROTEAN TGX precast gels, and transferred onto nitrocellulose membranes (Bio-Rad). The primary antibodies used are listed in Supplementary Table 7. For protein detection, the WesternBreeze Immunodetection System (Invitrogen) was used, and light emission was detected by either film exposure or the BioSpectrum Imaging system (UPV). Approximately 106 cells per sample were resuspended in PBS blocked using Fc blocker for 10 min on ice, followed by staining with the appropriate dilution of the antibodies or their respective isotype controls for 15 min on ice. Cells were washed and resuspended in PBS with propidium iodide (0.2 μg/ml) or DAPI (0.75 μg/ml) as a dead-cell marker. The antibodies used for flow cytometry are listed in Supplementary Table 7. For competitive-growth assays, the percentage of GFP+ cells was monitored by flow cytometry. For annexin V staining, annexin V binding buffer (BD Bioscience) was used instead of PBS and 7-aminoactinomycin D (7AAD; BD Bioscience) instead of propidium iodide. Phycoerythrin-labelled annexin V was purchased from BD Bioscience. For BrdU staining, the BrdU Flow Kit was purchased from BD Bioscience and used according to the manufacturer’s protocol. Methylcellulose colony-forming assays were performed with 10,000 BCR–ABL1 pre-B ALL cells. Cells were resuspended in mouse MethoCult medium (StemCell Technologies) and cultured on 3-cm dishes, with an extra water supply dish to prevent evaporation. Images were taken and colony numbers were counted after 14 days. Cell viability upon the genetic loss of function of target genes and/or inducible expression of PAX5 was monitored by flow cytometry using propidium iodide (0.2 μg/ml) as a dead-cell marker. To study the effects of an AMPK inhibitor (BML275), glucocorticoids (dexamethasone and prednisolone), CNR2 agonist (HU308), or TXNIP agonists (3-OMG and d-allose), 40,000 human or mouse leukaemia cells were seeded in a volume of 80 μl in complete growth medium on opaque-walled, white 96-well plates (BD Biosciences). Compounds were added at the indicated concentrations giving a total volume of 100 μl per well. After culturing for 3 days, cells were subjected to CellTiter-Glo Luminescent Cell Viability Assay (Promega). Relative viability was calculated using baseline values of cells treated with vehicle control as a reference. Combination index (CI) was calculated using the CalcuSyn software to determine interaction (synergistic, CI < 1; additive, CI = 1; or antagonistic, CI > 1) between the two agents. Constant ratio combination design was used. Concentrations of BML275, d-allose, 3-OMG and HU308 used are indicated in the figures. Concentrations of Dex used were tenfold lower than those of BML275. Concentrations of prednisolone used were twofold lower than those of BML275. To determine the number of viable cells, the trypan blue exclusion method was applied, using the Vi-CELL Cell Counter (Beckman Coulter). ChIP was performed as described previously33. Chromatin from fixed patient-derived Ph+ ALL cells (ICN1) was isolated and sonicated to 100–500-bp DNA fragments. Chromatin fragments were immunoprecipitated with either IgG (as a control) or anti-Pax5 antibody (see Supplementary Table 7). Following reversal of crosslinking by formaldehyde, specific DNA sequences were analysed by quantitative real-time PCR (see Supplementary Table 8 for primers). Primers were designed according to ChIP–seq tracks for PAX5 antibodies in B lymphocytes (ENCODE, Encyclopedia of DNA Elements, GM12878). ChIP–seq tracks for PAX5, IKZF1, EBF1 and TCF3 antibodies in a normal B-cell sample (ENCODE GM12878, UCSC genome browser) on INSR, GLUT1, GLUT3, GLUT6, HK2, G6PD, NR3C1, TXNIP, CNR2 and LKB1 gene promoter regions are shown. CD19 and ACTA1 served as a positive and a negative control gene, respectively. The y axis represents the normalized number of reads per million reads for peak summit for each track. The ChIP–seq peaks were called by the MACS peak-caller by comparing read density in the ChIP experiment relative to the input chromatin control reads, and are shown as bars under each wiggle track. Gene models are shown in UCSC genome browser hg19. Extracellular glucose levels were measured using the Amplex Red Glucose/Glucose Oxidase Assay Kit (Invitrogen), according to the manufacturer’s protocol. Glucose concentrations were measured in fresh and spent medium. Total ATP levels were measured using the ATP Bioluminescence Assay Kit CLS II (Roche) according to the manufacturer’s protocol. In fresh medium, 1 × 106 cells per ml were seeded and treated as indicated in the figure legends. Relative levels of glucose consumed and total ATP are shown. All values were normalized to cell numbers (Figs 1b, c, 2c (glucose uptake), 3a and Extended Data Figs 2c, 4f, 6d) or total protein (Fig. 2c, ATP levels). Numbers of viable cells were determined by applying trypan blue dye exclusion, using the Vi-CELL Cell Counter (Beckman Coulter). Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured using a Seahorse XFe24 Flux Analyzer with an XF Cell Mito Stress Test Kit and XF Glycolysis Stress Test Kit (Seahorse Bioscience) according to the manufacturer’s instructions. All compounds and materials were obtained from Seahorse Bioscience. In brief, 1.5 × 105 cells per well were plated using Cell-Tak (BD Biosciences). Following incubation in XF-Base medium supplemented with glucose and GlutaMAX for 1 h at 37 °C (non-CO incubator) for pH stabilization, OCR was measured at the resting stage (basal respiration in XF Base medium supplemented with GlutaMax and glucose) and in response to oligomycin (1 μmol/l; mitochondrial ATP production), mitochondrial uncoupler FCCP (5 μmol/l; maximal respiration), and respiratory chain inhibitor antimycin and rotenone (1 μmol/l). Spare respiratory capacity is the difference between maximal respiration and basal respiration. ECAR was measured under specific conditions to generate glycolytic profiles. Following incubation in glucose-free XF Base medium supplemented with GlutaMAX for 1 h at 37 °C (non-CO incubator) for pH stabilization, basal ECAR was measured. Following measurement of the glucose-deprived, basal ECAR, changes in ECAR upon the sequential addition of glucose (10 mmol/l; glycolysis), oligomycin (1 μmol/l; glycolytic capacity), and 2-deoxyglucose (0.1 mol/l) were measured. Glycolytic reserve was determined as the difference between oligomycin-stimulated glycolytic capacity and glucose-stimulated glycolysis. All values were normalized to cell numbers (Extended Data Fig. 2c) or total protein (Extended Data Figs 3a, 7a, b 8f) and are shown as the fold change relative to basal ECAR or OCR. Metabolite extraction and mass-spectrometry-based analysis were performed as described previously34. Metabolites were extracted from 2 × 105 cells per sample using the methanol/water/chloroform method. After incubation at 37 °C for the indicated time, cells were rinsed with 150 mM ammonium acetate (pH 7.3), and 400 μl cold 100% methanol (Optima* LC/MS, Fisher) and then 400 μl cold water (HPLC-Grade, Fisher) was added to cells. A total of 10 nmol norvaline (Sigma) was added as internal control, followed by 400 μl cold chloroform (HPLC-Grade, Fisher). Samples were vortexed three times over 15 min and spun down at top speed for 5 min at 4 °C. The top layer (aqueous phase) was transferred to a new Eppendorf tube, and samples were dried on Vacufuge Plus (Eppendorf) at 30 °C. Extracted metabolites were stored at −80 °C. For mass spectrometry-based analysis, the metabolites were resuspended in 70% acetonitrile and 5 μl used for analysis with a mass spectrometer. The mass spectrometer (Q Exactive, Thermo Scientific) was coupled to an UltiMate3000 RSLCnano HPLC. The chromatography was performed with 5 mM NH AcO (pH 9.9) and acetonitrile at a flow rate of 300 μl/min starting at 85% acetonitrile, going to 5% acetonitrile at 18 min, followed by an isocratic step to 27 min and re-equilibration to 34 min. The separation was achieved on a Luna 3u NH2 100A (150 × 2 mm) (Phenomenex). The Q Exactive was run in polarity switching mode (+3 kV/−2.25 kV). Metabolites were detected based on retention time (t ) and on accurate mass (± 3 p.p.m.). Metabolite quantification was performed as area-under-the-curve (AUC) with TraceFinder 3.1 (Thermo Scientific). Data analysis was performed in R (https://www.r-project.org/), and data were normalized to the number of cells. Relative amounts were log -transformed, median-centred and are shown as a heat map. To generate a model for pre-leukaemic B cell precursors expressing BCR–ABL1, BCR–ABL1 knock-in mice were crossed with Mb1-Cre deleter strain (Mb1-Cre; Bcr+/LSL-BCR/ABL) for excision of a stop-cassette in early pre-B cells. Bone marrow cells collected from Mb1-Cre; Bcr+/LSL-BCR/ABL mice cultured in the presence of IL-7 were primed with vehicle control or a combination of OAA (8 mmol/l), DMS (8 mmol/l) and insulin (210 pmol/l). Following a week of priming, cells were maintained and expanded in the presence of IL-7, supplemented with vehicle control or a combination of OAA (0.8 mmol/l) and DMS (0.8 mmol/l) for 4 weeks. Pre-B cells from Mb1-Cre; Bcr+/LSL-BCR/ABL mice expressed low levels of BCR–ABL1 tagged to GFP, and were analysed by flow cytometry for surface expression of GFP and CD19. The methylcellulose colony-forming assays were performed with 10,000 cells treated with vehicle control or metabolites. Cells were resuspended in mouse MethoCult medium (StemCell Technologies) and cultured on 3-cm diameter dishes, with an extra water supply dish to prevent evaporation. Images were taken and colony numbers counted after 14 days. For in vivo transplantation experiments, cells were treated with vehicle control or metabolites (OAA/DMS) for 6 weeks. One million cells were intravenously injected into sublethally irradiated (250 cGy) 6–8-week-old female NSG mice (n = 7 per group). Mice were randomly allocated into each group, and the minimal number of mice in each group was calculated by using the ‘cpower’ function in R/Hmisc package. No blinding was used. Each mouse was killed when it became terminally sick and showed signs of leukaemia burden (hunched back, weight loss and inability to move). The bone marrow and spleen were collected for flow cytometry analyses for leukaemia infiltration (CD19, B220). After 63 days, all remaining mice were killed and bone marrow and spleens from all mice were analysed by flow cytometry. Statistical analysis was performed using the Mantel–Cox log-rank test. All mouse experiments were in compliance with institutional approval by the University of California, San Francisco Institutional Animal Care and Use Committee. Following cytokine-independent proliferation, BCR–ABL1-transformed Lkb1fl/fl or AMPKa2fl/fl pre-B ALL cells were transduced with 4-OHT-inducible Cre or an empty vector control. For ex vivo deletion, deletion was induced 24 h before injection. For in vivo deletion, deletion was induced by 4-OHT (0.4 mg per mouse; intraperitoneal injection). Approximately 106 cells were injected into each sublethally irradiated (250 cGy) NOD/SCID mouse. Seven mice per group were injected via the tail vein. We randomly allocated 6–8-week-old female NOD/SCID or NSG mice into each group. The minimal number of mice in each group was calculated using the ‘cpower’ function in R/Hmisc package. No blinding was used. When a mouse became terminally sick and showed signs of leukaemia burden (hunched back, weight loss and inability to move), it was killed and the bone marrow and/or spleen were collected for flow cytometry analyses for leukaemia infiltration. Statistical analysis was performed by Mantel–Cox log-rank test. In vivo expansion and leukaemia burden were monitored by luciferase bioimaging. Bioimaging of leukaemia progression in mice was performed at the indicated time points using an in vivo IVIS 100 bioluminescence/optical imaging system (Xenogen). d-luciferin (Promega) dissolved in PBS was injected intraperitoneally at a dose of 2.5 mg per mouse 15 min before measuring the luminescence signal. General anaesthesia was induced with 5% isoflurane and continued during the procedure with 2% isoflurane introduced through a nose cone. All mouse experiments were in compliance with institutional approval by the University of California, San Francisco Institutional Animal Care and Use Committee. Data are shown as mean ± s.d. unless stated. Statistical significance was analysed by using Grahpad Prism software or R software (https://www.r-project.org/) by using two-tailed t-test, two-way ANOVA, or log-rank test as indicated in figure legends. Significance was considered at P < 0.05. For in vitro experiments, no statistical methods were used to predetermine the sample size. For in vivo transplantation experiments, the minimal number of mice in each group was calculated through use of the ‘cpower’ function in the R/Hmisc package. No animals were excluded. Overall survival and relapse-free survival data were obtained from GEO accession number GSE11877 (refs 35, 36) and TCGA. Kaplan–Meier survival analysis was used to estimate overall survival and relapse-free survival. Patients with high risk pre-B ALL (COG clinical trial, P9906, n = 207; Supplementary Table 10) were segregated into two groups on the basis of high or low mRNA levels with respect to the median mRNA values of the probe sets for the gene of interest. A log-rank test was used to compare survival differences between patient groups. R package ‘survival’ Version 2.35-8 was used for the survival analysis and Cox proportional hazards regression model in R package for the multivariate analysis (https://www.r-project.org/). The investigators were not blinded to allocation during experiments and outcome assessment. Experiments were repeated to ensure reproducibility of the observations. Gel scans are provided in Supplementary Fig. 1. Gene expression data were obtained from the GEO database accession numbers GSE32330 (ref. 12), GSE52870 (ref. 37), and GSE38463 (ref. 38). Patient-outcome data were derived from the National Cancer Institute TARGET Data Matrix of the Children’s Oncology Group (COG) Clinical Trial P9906 (GSE11877)35, 36 and from TCGA (the Cancer Genome Atlas). GEO accession details are provided in Supplementary Tables 9 and 10. ChIP–seq tracks for PAX5, IKZF1, EBF1 and TCF3 antibodies in a normal B-cell sample (ENCODE GM12878, UCSC genome browser) on INSR, GLUT1, GLUT3, GLUT6, HK2, G6PD, NR3C1, TXNIP, CNR2 and LKB1 gene promoter regions are shown in UCSC genome browser hg19. All other data are available from the corresponding author upon reasonable request.


News Article | March 1, 2017
Site: www.chromatographytechniques.com

Phenomenex’s two additions to its BioSolutions portfolio offer high-efficiency reversed-phase characterization of synthetic DNA and RNA. The Clarity Oligo-XT C18 columns feature novel and robust core-shell media and increased sensitivity that improves quantitation by mass spectrometry. The core-shell particles deliver the separation power necessary to accurately resolve closely related synthetic oligonucleotide sequences. The columns are available in directly scalable 1.7, 2.6 and 5µm particle sizes that enable easy method transfer between analytical HPLC/UHPLC instrumentation and preparative purifications systems. The Clarity Oligo-SAX columns feature an entirely new, rugged non-porous particle that retains synthetic oligonucleotides through strong ion exchange mechanisms, adding a robust strong anion exchanger choice with improved column lifetimes to the Clarity family. These quaternary amine functionalized, nonporous particles are engineered for performance at high pH (2.5 to 12.5) and temperatures up to 85C and are provided in 5µm particle size for analytical characterization. Phenomenex, Inc. www.phenomenex.com, 310-212-0555


News Article | February 24, 2017
Site: www.prnewswire.co.uk

According to a new market research report "High-performance Liquid Chromatography (HPLC) Market by Product (Instruments (Systems, Detectors), Consumables (Columns, Filters), and Accessories), Application (Clinical Research, Diagnostics, Forensics) - Analysis & Global Forecast to 2021", published by MarketsandMarkets, This report studies the global HPLC Market for the forecast period of 2016 to 2021. This market is expected to reach 4.13 Billion by 2021 from USD 3.23 Billion in 2016, growing at a CAGR of 5.1%. Browse 112 market data Tables and 42 Figures spread through 182 Pages and in-depth TOC on "High-performance Liquid Chromatography (HPLC) Market" Early buyers will receive 10% customization on this report. The global HPLC Market is segmented on the basis of product, application, and region. On the basis application, the HPLC is segmented into clinical research, diagnostics, forensics, and other applications (including food & environmental analysis and academic research). In 2016, the clinical research segment is expected to account for the largest share of the global HPLC Market. On the basis of product, the HPLC Market is categorized into instruments, consumables, and accessories. The instruments segment is estimated to account for the largest share of the global HPLC Market, by product. The consumables segment is projected to grow at the highest CAGR between 2016 and 2021, primarily due to the recurring requirement of consumables. The instruments segment is further categorized into systems, detectors, pumps, and fraction collectors. In 2016, the systems segment is expected to command the largest share and the highest growth of the instruments market. The consumables segment is further categorized into columns, filters, vials, and tubes. The columns segment is estimated to grow at the highest CAGR during the forecast period. This segment is further categorized into reverse-phase HPLC columns, normal-phase/hydrophobic interaction HPLC columns, ion exchange HPLC columns, and other columns. Based on region, the HPLC Market is divided into North America, Europe, Asia-Pacific, and the Rest of the World (RoW). The RoW region comprises Latin America, the Middle East, and Africa. In 2016, North America is projected to account for the largest share of the HPLC Market, followed by Europe and Asia-Pacific. Increasing funding for R&D, preclinical activities by CROs and pharmaceutical companies, and the growing food industry in Ontario are propelling the growth of the North American HPLC Market. The major players in the global HPLC Market are Waters Corporation (U.S.), Agilent Technologies (U.S.), and Shimadzu Corporation (Japan). These companies are dominant in the HPLC Market mainly due to their well-established presence in the field of chromatography, presence in over 50 countries, high R&D investments, and strong sales and distribution force. The other players in the market include Thermo Fisher Scientific Inc. (U.S.), GE Healthcare (U.S.), PerkinElmer, Inc. (U.S.), Bio-Rad Laboratories, Inc. (U.S.), Gilson, Inc. (U.S.), Phenomenex, Inc. (U.S.), and JASCO, Inc. (U.S.). PREPARATIVE AND PROCESS CHROMATOGRAPHY MARKET By Type (Preparative, Process), Products (Systems, Columns, Empty, Glass, Resins, Protein A, Affinity, Ion exchange, Mixed mode, Services), End User (Biotechnology, Pharmaceutical) - Global Forecasts to 2021. CHROMATOGRAPHY INSTRUMENTS MARKET By System (LC (HPLC, UHPLC, Flash), GC, Other Components (Autosamplers, Detectors, Fraction Collectors), Consumable (Reverse Phase Columns, Syringe Filters, Vials) - Analysis & Global Forecasts to 2020. MarketsandMarkets is the largest market research firm worldwide in terms of annually published premium market research reports. Serving 1700 global fortune enterprises with more than 1200 premium studies in a year, M&M is catering to a multitude of clients across 8 different industrial verticals. We specialize in consulting assignments and business research across high growth markets, cutting edge technologies and newer applications. Our 850 fulltime analyst and SMEs at MarketsandMarkets are tracking global high growth markets following the "Growth Engagement Model - GEM". The GEM aims at proactive collaboration with the clients to identify new opportunities, identify most important customers, write "Attack, avoid and defend" strategies, identify sources of incremental revenues for both the company and its competitors. M&M's flagship competitive intelligence and market research platform, "RT" connects over 200,000 markets and entire value chains for deeper understanding of the unmet insights along with market sizing and forecasts of niche markets. The new included chapters on Methodology and Benchmarking presented with high quality analytical infographics in our reports gives complete visibility of how the numbers have been arrived and defend the accuracy of the numbers. We at MarketsandMarkets are inspired to help our clients grow by providing apt business insight with our huge market intelligence repository. Connect with us on LinkedIn @ http://www.linkedin.com/company/marketsandmarkets

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