Neutrophils were harvested and purified from bone marrow as described above, and plated at 5×106 cells/mL in cRPMI with 10ng/mL TNFα. After 12h, cells were restimulated for 30 min with Pam3CSK4 or fMLP. Eicosanoids in neutrophils and secreted into the culture medium were quantified via liquid chromatography-tandem mass spectrometry (LC-MS/MS) as described before (18 (link)–20 (link)). In brief, cold methanol was added to cultured neutrophils in media to stabilize lipid mediators. Deuterated internal standards (PGE2-d4, LTB4-d4, 15-HETE-d8, LXA4-d5, DHA-d5, AA-d8) were added to samples to calculate extraction recovery. LC/MS/MS system consisted of an Agilent 1200 Series HPLC, Kinetex C18 minibore column (Phenomenex, Torrance, CA, USA), and AB Sciex QTRAP 3200 mass spectrometer. Analysis was carried out in negative ion mode, and lipid mediators quantitated using scheduled multiple reaction monitoring (MRM) mode using specific and established transition ions.
Kinetex c18 minibore column
Manufactured by Phenomenex
Sourced in United States
The Kinetex C18 minibore column is a high-performance liquid chromatography (HPLC) column designed for analytical separations. It features a 2.6 μm core-shell particle technology that provides efficient and fast chromatographic separations. The column dimensions are 100 mm x 2.1 mm, making it suitable for analytical applications requiring smaller sample volumes.
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Lab products found in correlation
2 protocols using kinetex c18 minibore column
1
Neutrophil Eicosanoid Quantification
2
Lipidomic Analysis of Experimental Autoimmune Uveitis
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Whole lymph nodes and eye globes were harvested at disease onset and peak inflammation from mice induced for EAU by immunization with IRBP651-670 peptide. Tissues from naïve mice served as healthy controls. Tissues were homogenized in 66% MeOH containing deuterated internal standards with Bead Ruptor, and lipid mediators were extracted using solid phase C-18 columns. Mouse serum was combined with 2 volumes of ice cold MeOH containing deuterated internal standards. Diluted serum samples were vortexed and incubated at −80°C for 1 hr to precipitate proteins. Supernatants were collected and extracted using solid phase C-18 columns (Gao et al., 2018 (link)). Lipid mediators were then quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Extraction recovery was calculated based on deuterated internal standards (PGE2-d4, LTB4-d4, 15-HETE-d8, LXA4-d5, DHA-d5, AA-d8). The LC-MS/MS system was composed of Agilent 1200 series HPLC, Kinetex C18 minibore column (Phenomenex, CA), and AB Sciex QTRAP 4500 mass spectrometer (SCIEX, MA). Analyses were performed in negative ion mode with scheduled multiple reaction monitoring using 4–5 transition ions per lipid mediator (Livne-Bar et al., 2017 (link); von Moltke et al., 2012 (link)).
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