Strains were grown in THY, THY-Tween 80, THY-Plasma or THY-17:1 until OD600nm = 0.4–0.5. Fatty acids were extracted and analyzed as previously described [2 (link), 3 (link), 5 (link)]. Briefly, analyses were performed in a split-splitless injection mode on an AutoSystem XL Gas Chromatograph (Perkin-Elmer) equipped with a ZB-Wax capillary column (30 m x 0.25 mm x 0.25 mm; Phenomenex, France). Data were recorded and analyzed by TotalChrom Workstation (Perkin-Elmer). FA peaks were detected between 12 and 40 min of elution, and identified by comparing to retention times of purified esterified FA standards (Mixture ME100, Larodan, Sweden). Results are shown as percent of specific FA as calculated from their proportions compared to total peak areas (TotalChrom Workstation; Perkin Elmer).
Totalchrom workstation
Manufactured by PerkinElmer
Sourced in United States
The TotalChrom Workstation is a software solution designed for chromatography data analysis and reporting. It provides a comprehensive platform for managing and processing chromatographic data from various instruments. The TotalChrom Workstation enables users to acquire, analyze, and report on chromatographic data in a consistent and efficient manner.
Automatically generated - may contain errors
Lab products found in correlation
Autosystem xl gas chromatograph, by PerkinElmer (2 mentions)
Zb wax capillary column, by Phenomenex (2 mentions)
Me100, by Larodan (1 mentions)
Methyl 10 undecenoate, by Merck Group (1 mentions)
Myo 2 3h inositol, by PerkinElmer (1 mentions)
Series 200 hplc system, by PerkinElmer (1 mentions)
Series 200, by PerkinElmer (1 mentions)
Luna c8, by Phenomenex (1 mentions)
Hypersil bds c8, by Thermo Fisher Scientific (1 mentions)
Autosystem xl, by PerkinElmer (1 mentions)
5 protocols using totalchrom workstation
1
Fatty Acid Profiling in Bacterial Strains
2
Quantification of Bacterial Fatty Acids
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Whole cell esterified fatty acid determinations were done essentially as described62 (link). Briefly, 1–2 ml S. aureus cultures (OD600=≥1) were centrifuged, washed once in 0.9% NaCl containing 0.02% Triton X-100, then washed twice in 0.9% NaCl. Cell pellets were treated with 0.5 ml of 1 N sodium methoxide. Heptane (200 μl) was then added, together with methyl-10-undecenoate (Sigma-Aldrich) as internal standard, vortexed for 1 min, and centrifuged. Fatty acid methyl esters were recovered in the heptane phase. Analyses were performed in a split-splitless injection mode on an AutoSystem XL Gas Chromatograph (Perkin-Elmer) equipped with a ZB-Wax capillary column (30 m × 0.25 mm × 0.25 μm; Phenomenex, France). Data were recorded and analysed by TotalChrom Workstation (Perkin-Elmer). S. aureus fatty acid peaks were detected between 12 and 30 min of elution.
3
Quantification of Cellular Phosphoinositides
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Analysis of cellular phosphoinositide levels was performed as previously described (44 (link)) with the following modifications. Midlog cells (5 OD600 equivalents) grown in yeast nitrogen base media were harvested, washed in inositol free media (IFM), incubated for 10 min in inositol-free media, and labeled with myo-[2-3H] inositol (PerkinElmer) for 30 min. Following this initial labeling, cultures were split and further incubated in IFM or IFM containing 1.2 M NaCl for 15 min. Cells were precipitated in 4.5% perchloric acid and lysed by vortexing with glass beads. Cell lysates were washed in 100 mM EDTA, phospholipids were deacylated, and further processed as previously described (44 (link)). Samples were dried and resuspended in H2O. Identification and quantitation of 3H-labeled glycerolphosphoinositols was performed by HPLCy (HPLC) using a Series 200 HPLC system (PerkinElmer), a partisphere SAX column (HiChrom), an inflow 150TR radiomatic detector (PerkinElmer), TCNav software, and TotalChrom workstation (PerkinElmer). Data for individual phosphoinositide species data are shown as percentages of the total number of counts detected for normalization.
4
HPLC System for Analysis
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The HPLC system consisted of a quaternary pump (series 200, Perkin Elmer), auto sampler (series 200, Perkin Elmer), and temperature-controlled column oven (series 200, Perkin Elmer). The UV–VIS detector (series 200, Perkin Elmer) was operated at a wavelength of 229 nm. Data collection and analyses were performed using Totalchrom workstation, version 6.3.1.0504 (Perkin Elmer Inc., USA). Columns used were Luna C8, 100 × 4.6 mm, 3.0 μm (Phenomenex, USA), and Hypersil BDS C8, 100 × 4.6 mm, 3 μm (Thermo Scientific, UK).
5
EtOH Blood Concentration Determination
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From EtOH-treated rats, 100 μL of blood was collected and mixed with 500 μL of 0.015% propionitrile (internal standard). The ethanol level was determined using gas chromatography (GC) with the head-space technique on AutoSystem XL with Headspace Sampler TuboMatrix 40 (PerkinElmer, Boston, MA, USA) and TotalChrom Workstation Version 6.2.0. software, equipped with two parallel capillary columns Elite BAC-1, BAC-2 (Perkin Elmer, length: 30 m, diameter: 0.32 mm, film thicknesses: 1.8 mm and 1.2 mm) according to the Perkin Elmer’s instruction for EtOH blood level determination in the calibration range of 0.1–5 g/L.
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