6495 triple quad qqq ms

Manufactured by Agilent Technologies

The 6495 Triple Quad QQQ-MS is a high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) system designed for sensitive and precise quantitative analysis. It features a triple quadrupole mass analyzer configuration that provides reliable and reproducible results for a wide range of applications.

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Lab products found in correlation

Zorbax eclipse plus c18 column, by Agilent Technologies (6 mentions) 1290 infinity 2 uhplc, by Agilent Technologies (6 mentions) Masshunter software, by Agilent Technologies (4 mentions) Masshunter b 07, by Agilent Technologies (1 mentions)

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6495 QQQ-MS (8 citations)

6 protocols using 6495 triple quad qqq ms

Lipid Extraction and Quantification Protocol

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The protocol for lipid extraction was adapted from Matyash et al.^{48 (link)}. Frozen cell pellets (5 × 10⁵ cells) were resuspended in ice cold PBS and transferred to glass tubes before the addition of methanol and methyl tert-butyl ether. The tubes were then shaken for 1 h at 4 °C. Water was added to separate the phases before centrifugation at 1000 × g for 10 min. The upper organic phase was collected and dried in a Genevac EZ2 speed vac. Samples were resuspended in 2:1:1 isopropanol:acetonitrile:water prior to analysis. LC–MS was carried out using an Agilent Zorbax Eclipse Plus C18 column using an Agilent 1290 Infinity II UHPLC inline with an Agilent 6495 Triple Quad QQQ-MS. Lipids were identified by fragmentation and retention time, and comparison to standards, and were quantified using Agilent Mass Hunter software. Comparisons were made between relative amounts of lipid between conditions, extracted from equivalent cell numbers. Peak areas were quantile-normalized across the batch to generate the lipid intensities used for the plots and subsequent statistics shown in this manuscript.

Castoldi A., Monteiro L.B., van Teijlingen Bakker N., Sanin D.E., Rana N., Corrado M., Cameron A.M., Hässler F., Matsushita M., Caputa G., Klein Geltink R.I., Büscher J., Edwards-Hicks J., Pearce E.L, & Pearce E.J. (2020). Triacylglycerol synthesis enhances macrophage inflammatory function. Nature Communications, 11, 4107.

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Quantitative Lipid Extraction and Analysis

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Lipids were extracted using a biphasic methyl tert-butyl ether (MTBE) extraction protocol. The same number of cells per condition were resuspended in 100 μl cold PBS in glass vials. Cold methanol (750 μl), MTBE (2 ml) and water (625 μl) were added sequentially with vortexing. Samples were centrifuged to separate phases, and the upper organic phase was dried using a Genevac EZ2 speed vac. Samples were resuspended in 2:1:1 isopropanol: acetonitrile: water prior to analysis. LC–MS was carried out using an Agilent Zorbax Eclipse Plus C18 column using an Agilent 1290 Infinity II UHPLC in line with an Agilent 6495 Triple Quad QQQ-MS. Lipids were identified by fragmentation and retention time, and quantified using MassHunter B.07.01 (Agilent).

Baixauli F., Piletic K., Puleston D.J., Villa M., Field C.S., Flachsmann L.J., Quintana A., Rana N., Edwards-Hicks J., Matsushita M., Stanczak M.A., Grzes K.M., Kabat A.M., Fabri M., Caputa G., Kelly B., Corrado M., Musa Y., Duda K.J., Mittler G., O’Sullivan D., Sesaki H., Jenuwein T., Buescher J.M., Pearce E.J., Sanin D.E, & Pearce E.L. (2022). An LKB1–mitochondria axis controls TH17 effector function. Nature, 610(7932), 555-561.

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Lipid Extraction and LC-MS Analysis Protocol

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The protocol for lipid extraction was adapted from Matyash et al. (Matyash et al., 2008 (link)). Frozen cell pellets (60000-150000 cells) were resuspended in ice cold PBS and transferred to glass tubes before the addition of methanol and methyl tert-butyl ether. The tubes were then shaken for 1 h at 4°C. Water was added to separate the phases before centrifugation at 1,000 x g for 10 min. The upper organic phase was collected and dried in a Genevac EZ2 speed vac. Samples were resuspended in 2:1:1 isopropanol:acetonitrile:- milli-Q H2O priorto analysis. LC-MS was carried out using an Agilent Zorbax Eclipse Plus C18 column using an Agilent 1290 Infinity II UHPLC inline with an Agilent 6495 Triple Quad QQQ-MS. Lipids were identified by fragmentation and retention time, and were quantified using Agilent Mass Hunter software.

Remmerie A., Martens L., Thoné T., Castoldi A., Seurinck R., Pavie B., Roels J., Vanneste B., De Prijck S., Vanhockerhout M., Binte Abdul Latib M., Devisscher L., Hoorens A., Bonnardel J., Vandamme N., Kremer A., Borghgraef P., Van Vlierberghe H., Lippens S., Pearce E., Saeys Y, & Scott C.L. (2020). Osteopontin Expression Identifies a Subset of Recruited Macrophages Distinct from Kupffer Cells in the Fatty Liver. Immunity, 53(3), 641-657.e14.

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Lipid Extraction and Quantification Protocol

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The protocol for lipid extraction was adapted from Matyash et al.⁵⁴ (link) Frozen cell pellets (5 × 10⁵ cells) were resuspended in ice-cold PBS and transferred to glass tubes before the addition of methanol and methyl tert-butyl ether. The tubes were then shaken for 1 h at 4°C. Water was added to separate the phases before centrifugation at 1000g for 10 min. The upper organic phase was collected and dried in a Genevac EZ2 speed vac. Samples were resuspended in 2:1:1 isopropanol: acetonitrile:water prior to analysis. LC-MS was carried out using an Agilent Zorbax Eclipse Plus C18 column using an Agilent 1290 Infinity II UHPLC in line with an Agilent 6495 Triple Quad QQQ-MS. Lipids were identified by fragmentation and retention time, and comparison to standards, and were quantified using Agilent Mass Hunter software. Comparisons were made between relative amounts of lipid between conditions, extracted from equivalent cell numbers. Peak areas were quantile-normalized across the batch to generate the lipid intensities used for the plots and subsequent statistics shown in this manuscript.

Castoldi A., Sanin D.E., van Teijlingen Bakker N., Aguiar C.F., de Brito Monteiro L., Rana N., Grzes K.M., Kabat A.M., Curtis J., Cameron A.M., Caputa G., Antônio de Souza T., Souto F.O., Buescher J.M., Edwards-Hicks J., Pearce E.L., Pearce E.J, & Saraiva Camara N.O. (2023). Metabolic and functional remodeling of colonic macrophages in response to high-fat diet-induced obesity. iScience, 26(10), 107719.

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Lipid Extraction and Quantification Protocol

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The protocol for lipid extraction was adapted from Matyash et al. (Matyash et al., 2008 (link)). Frozen cell pellets (60000-150000 cells) were resuspended in ice cold PBS and transferred to glass tubes before the addition of methanol and methyl tert-butyl ether. The tubes were then shaken for 1 h at 4°C. Water was added to separate the phases before centrifugation at 1,000 x g for 10 min. The upper organic phase was collected and dried in a Genevac EZ2 speed vac. Samples were resuspended in 2:1:1 isopropanol:acetonitrile:milli-Q H2O prior to analysis. LC-MS was carried out using an Agilent Zorbax Eclipse Plus C18 column using an Agilent 1290 Infinity II UHPLC inline with an Agilent 6495 Triple Quad QQQ-MS. Lipids were identified by fragmentation and retention time, and were quantified using Agilent Mass Hunter software.

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Lipid Extraction and Quantification Protocol

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Lipids were extracted using a biphasic Methyl tert-butyl ether (MTBE) extraction protocol (adapted from Matyash et al., 2008 (link)). In brief, cells were resuspended in 100uL cold PBS in glass vials. Cold methanol (750uL), MTBE (2mL), and water (625uL) were added sequentially with vortexing. Samples were centrifuged to separate phases, and the upper organic phase was dried using a Genevac EZ2 speed vac. Samples were resuspended in 2:1:1 isopropanol:acetonitrile:water prior to analysis. LC-MS was carried out using an Agilent Zorbax Eclipse Plus C18 column using an Agilent 1290 Infinity II UHPLC in line with an Agilent 6495 Triple Quad QQQ-MS. Lipids were identified by fragmentation and retention time, and were quantified using Agilent Mass Hunter software.

Corrado M., Edwards-Hicks J., Villa M., Flachsmann L.J., Sanin D.E., Jacobs M., Baixauli F., Stanczak M., Anderson E., Azuma M., Quintana A., Curtis J.D., Clapes T., Grzes K.M., Kabat A.M., Kyle R., Patterson A.E., Geltink R.K., Amulic B., Steward C.G., Strathdee D., Trompouki E., O’Sullivan D., Pearce E.J, & Pearce E.L. (2020). Dynamic Cardiolipin Synthesis Is Required for CD8+ T Cell Immunity. Cell Metabolism, 32(6), 981-995.e7.

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