PCTR1 (16R-glutathionyl, 17S-hydroxy-4Z,7Z,10Z,12E,14E,19Z-docosahexaenoic acid), 13 N-PCTR1, and 13 N-MCTR3 were each synthesized by total organic synthesis and provided by Dr. Nicos A. Petasis (University of Southern California) via subcontract for P01GM095467 to CNS. PCTR1, PD1 (10R,17S-dihydroxy-4Z,7Z,11E,13E,15Z,19Z-docosahexaenoic acid), 17-HDHA, d4-LTB4, d8-5S-HETE, d5-LTC4, d5-LTD4, LTC4, LTD4, and LTE4 were purchased from Cayman Chemical (Ann Arbor, MI). For cellular administration, PCTR1, PD1, or vehicle (ethanol) was diluted in media to a final ethanol concentration <0.1%. For mouse administration, PCTR1, PD1, or vehicle (ethanol) was brought to dryness with a gentle stream of nitrogen gas prior to resuspension in PBS. Before experiments, each SPM was authenticated and validated using an unbiased library search (Sciex OS Version 1.7.0.36606) and was in accordance with its reported physical properties established earlier (4 (link), 5 (link)).
D8 5s hete
Manufactured by Cayman Chemical
Sourced in Germany
D8-5S-HETE is a chemical compound produced by Cayman Chemical. It is a fatty acid metabolite that can be used for research purposes.
Automatically generated - may contain errors
Lab products found in correlation
Ltb4 d4, by Cayman Chemical (5 mentions)
Pge2 d4, by Cayman Chemical (4 mentions)
D5 lxa4, by Cayman Chemical (4 mentions)
Aa d8, by Cayman Chemical (3 mentions)
D5 rvd2, by Cayman Chemical (3 mentions)
17 hdha, by Cayman Chemical (1 mentions)
Dc protein assay reagents package, by Bio-Rad (1 mentions)
Sep pak vac 6 cc 500 mg 6 ml c18, by Waters Corporation (1 mentions)
Turbovap lv, by Biotage (1 mentions)
5 protocols using d8 5s hete
1
Synthesis and Characterization of Specialized Pro-resolving Lipid Mediators
2
Macrophage-E. coli Interaction Lipidome
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Macrophages (2x106 cells) were incubated with E. coli (1x107 cells) for 30 minutes at 37°C. Incubations were quenched using two volumes of ice-cold methanol containing deuterium labelled internal standards (500 pg each of d4-PGE2, d5-LXA4, d4-RvD2, d4-LTB4 and d8-5S-HETE, Cayman Chemicals). Samples were then stored at -20°C prior to extraction and lipid mediator profiling.
3
Lipid Mediator Extraction and Analysis
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Freshly isolated PMNL and monocytes as well as polarized MDMs and HEK293 cells (1 or 2 × 106 in 1 mL, as indicated) were incubated in PG buffer containing 1 mm CaCl2. In some experiments, cells were incubated in PG buffer containing 0.5 mm EDTA or 0.5 mm EDTA plus BAPTA/AM (20 µm ). AKBA and other compounds, E. coli (O6:K2:H1; ratio 1:50) or vehicle (0.1% DMSO) as well as AA, EPA, and DHA were added at 37 °C as indicated. After the indicated (pre‐)incubation times, the supernatants (1 mL) were mixed with 2 mL of ice‐cold methanol that contained deuterium‐labeled internal standards (200 nm d4‐PGE2, d4‐LTB4, d5‐LXA4, d5‐RvD2, and d8‐5S‐HETE, as well as 10 µm d8‐AA; Cayman Chemical/Biomol GmbH, Hamburg, Germany). Solid phase extraction (SPE) of LM, sample preparation, and UPLC‐MS‐MS analysis of LM was performed as described by Jordan et al.[15 ]
4
Quantification of Lipid Mediators in Monocytes and MDMs
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Monocytes and MDM subsets (2 × 106/mL) were incubated in 1 mL PBS containing 1 mM CaCl2 with E. coli (O6:K2:H1; ratio = 1:50) or 0.5% ECM at 37 °C. After the indicated incubation periods, the supernatants (1 mL) were transferred to 2 mL of ice-cold methanol containing 10 µL of deuterium-labeled internal standards (200 nM d8-5S-HETE, d4-LTB4, d5-LXA4, d5-RvD2, d4-PGE2 and 10 µM d8-AA; Cayman Chemical/Biomol GmbH, Hamburg, Germany) to facilitate LM quantification and sample recovery. Solid phase extraction of LM, sample preparation, and UPLC-MS-MS analysis of LM was conducted exactly as reported in Jordan et al. (43 (link)).
To consider reduced numbers of adherent MDM after GC-treatment (17 (link)) during stimulation with E. coli for LM analysis, the total protein amount was determined for all samples using a modified Lowry Protein Assay employing DC Protein Assay Reagents Package (#5000116, Bio Rad, Hercules, CA). LM formation, given in pg, was normalized to total protein amount adjusted to 0.15 mg (corresponding to the mean protein amount of 2 × 106 plated MDM 48 h prior determination) or 1 mg, as indicated.
To consider reduced numbers of adherent MDM after GC-treatment (17 (link)) during stimulation with E. coli for LM analysis, the total protein amount was determined for all samples using a modified Lowry Protein Assay employing DC Protein Assay Reagents Package (#5000116, Bio Rad, Hercules, CA). LM formation, given in pg, was normalized to total protein amount adjusted to 0.15 mg (corresponding to the mean protein amount of 2 × 106 plated MDM 48 h prior determination) or 1 mg, as indicated.
5
Extraction and Quantification of Lipid Mediators
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LMs were extracted from cell culture supernatants as described [26 (link)]. In brief, supernatants were transferred to ice-cold methanol (supernatant/methanol = 40/60) containing deuterium-labeled internal standards (200 nM d8-5S-HETE, d4-LTB4, d5-LXA4, d5-RvD2, d4-PGE2, and 10 μM d8-AA; Cayman Chemical/Biomol, Hamburg, Germany). After protein precipitation at −20 °C, samples were centrifuged, acidified (pH 3.5) and subjected to solid phase extraction (Sep-Pak® Vac 6 cc 500 mg/6 mL C18; Waters, Milford, USA). The cartridges were successively washed with methanol and n-hexane, and LMs were eluted with methyl formiate. Eluates were evaporated to dryness (TurboVap LV, Biotage, Uppsala, Sweden) and LMs taken up in methanol/water (50/50) for UPLC-MS/MS analysis.
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