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Sil 20ac ht

Manufactured by Thermo Fisher Scientific

The SIL-20AC HT is a high-temperature autosampler designed for use in analytical instrumentation. It is capable of operating at temperatures up to 800°C, making it suitable for a variety of high-temperature applications. The autosampler provides automated sample handling and injection, enabling efficient and consistent sample processing.

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6 protocols using sil 20ac ht

1

Lipid Quantification by HPLC-MS/MS

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Lipids were extracted from 50 μl of liver homogenate with an internal standard mixture. The extracted free fatty acids were derivatized by amino methyl phenyl pyridium (AMPP) into FA-AMPP derivatives in order to obtain high sensitivity in MS. Measurement of lipids was performed with a Shimadzu 10A HPLC system and a Shimadzu SIL-20AC HT auto-sampler coupled to a Thermo Scientific TSQ Quantum Ultra triple quadrupole mass spectrometer operated in SRM mode under ESI(+). Data processing was conducted with Xcalibur (Thermo).
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2

Cholesterol Quantification in Lysosomes

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The lysosomal samples were extracted from anti-HA magnetic beads with methanol and ethyl acetate with d7-cholesterol (2 μg per sample), and further derivatized with nicotinic acid to improve the mass spectrometric detection sensitivity of cholesterol. Measurement of cholesterol was performed with a Shimadzu 10A HPLC system and a Shimadzu SIL-20AC HT auto-sampler coupled to a Thermo Scientific TSQ Quantum Ultra triple quadrupole mass spectrometer. Data processing was conducted with Xcalibur™ Software version 4.0 (Thermo Fisher Scientific). A quality control (QC) sample was prepared by pooling the aliquots of the study samples and was used to monitor the instrument stability. The QC was injected six times in the beginning to stabilize the instrument and was injected every four study samples to monitor the instrument performance. The data was accepted if the coefficient variance (CV) of cholesterol in QC sample was < 15%. The data was reported as the peak area ratio of cholesterol to d7-cholesterol.
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3

Quantification of Diacylglycerol Lipids by LC-MS

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Diacylglycerol (DAG) and subspecies were quantitatively and qualitatively detected by LC-MS through Washington University Mass Spec Facility. In brief, lipids were extracted from 5×106 cells using a modified Bligh-Dyer method in the presence of an internal standard DG15:0-15:0 (0.5 µg per sample). Measurement of DAGs were performed with a Shimadzu 10A HPLC system and a Shimadzu SIL-20AC HT auto-sampler coupled to a Thermo Scientific TSQ Quantum Ultra triple quadrupole mass spectrometer operated in SRM mode under ESI(+). Data processing was conducted with X calibur (Thermo). Quality control (QC) samples were prepared by pooling the aliquots of study samples and were used to monitor instrument stability. The QC was injected seven times at the beginning to stabilize the instrument, and was injected again every five study samples. Only the lipid species with CV < 15% in QC sample were reported. Relative quantification of lipids was provided, and data were reported as the peak area ratios of the analytes to the internal standard. Relative quantification data generated in same batches are appropriate to compare the change of an analyte in AKR1B10 expression or AKR1B10 silencing samples to the corresponding control.
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4

Quantification of Lipid Profiles in Mouse Hearts

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The abundance of PA, DAG, triglyceride, and CL in mouse hearts was determined as described with modification (22 (link)). The LC-MS analysis was performed either with a Shimadzu 10A HPLC system and a Shimadzu SIL-20AC HT auto-sampler coupled to a Thermo Scientific TSQ Quantum Ultra triple quadrupole (TQ) mass spectrometer operated in selected reaction monitoring mode or with a Thermo Fisher Scientific Vantage TSQ mass spectrometer with Thermo Accela UPLC operated by Xcalibur software using selected ion monitoring mode. PA-(14:0)2, DAG-(15:0)2, triglyceride-(17:0)3, and CL-(14:0-14:0)2 were used as internal standards. Quantification of lipids was based on the ratio of the peak area of the analyte to the internal standard. For example, the ratio of CL-(18:2/18:2)2 and CL-(14:0-14:0)2 is used for measurement of CL-(18:2/18:2)2.
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5

Cholesterol Quantification in Lysosomes

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The lysosomal samples were extracted from anti-HA magnetic beads with methanol and ethyl acetate with d7-cholesterol (2 μg per sample), and further derivatized with nicotinic acid to improve the mass spectrometric detection sensitivity of cholesterol. Measurement of cholesterol was performed with a Shimadzu 10A HPLC system and a Shimadzu SIL-20AC HT auto-sampler coupled to a Thermo Scientific TSQ Quantum Ultra triple quadrupole mass spectrometer. Data processing was conducted with Xcalibur™ Software version 4.0 (Thermo Fisher Scientific). A quality control (QC) sample was prepared by pooling the aliquots of the study samples and was used to monitor the instrument stability. The QC was injected six times in the beginning to stabilize the instrument and was injected every four study samples to monitor the instrument performance. The data was accepted if the coefficient variance (CV) of cholesterol in QC sample was < 15%. The data was reported as the peak area ratio of cholesterol to d7-cholesterol.
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6

Quantification of Phosphatidic Acid in Liver and Plasma

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For the time course studies, PA was measured in liver and plasma using LC-ESI-MS/MS. Briefly, mouse liver samples were homogenized in 400 µL of water with the Omni Bead Ruptor 24 (Omni International, Inc., Kennesaw, GA). PA(14:0/14:0) was used as an internal standard (IS) and was added to the samples before extraction. Extraction of PA was performed by protein precipitation from 50 µL of plasma or liver homogenate. PA analysis was performed with a Shimadzu 10A HPLC system and a Shimadzu SIL-20AC HT auto-sampler coupled to a Thermo Scientific TSQ Quantum Ultra triple-quadrupole mass spectrometer operated in SRM mode under ESI(+). Data processing was performed using Xcalibur (Thermo, Waltham, MA). For each experiment, the data were reported as peak AUC ratios relative to the IS, and then normalized to the control group mean. All PA data for each experiment were collected at the same time, so that comparison of the ratios to the within-experiment controls allowed accurate relative quantification. For the FSG67 experiments, total PA (PA+lyso-PA) was measured using a kit from Cell Biolabs (San Diego, CA). All liver PA levels were normalized to protein measured using the BCA assay before expressing as fold over control.
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