Methyl-β-cyclodextrin (MβCD)-cholesterol complex commercially available (Sigma, C4951). For MβCD-sterol complexation, 50 mg/mL sterol stocks in 1:1 chloroform:methanol were prepared for desmosterol, lathosterol, cholestenone, cholesterol sulfate, 4β-hydroxycholesterol (Avanti Polar Lipids), 7DHC and cholesterol (Sigma or Avanti Polar Lipids). Stoichiometry of MβCD-cholesterol complexes typically consists of one cholesterol molecule entrapped within two CDs (1:2 molar ratio). We found optimal loading conditions using a 1:7 molar ratio, where desired sterol concentration was dried under nitrogen flow, followed by addition of 5 mM MβCD (Alfa Aesar, 1303.31 g/mol) prepared in serum-free DMEM. Sterol suspensions were sonicated for 5 min and incubated at 37°C with agitation overnight. Sterol crystals were subsequently removed via 0.45 μm filtration. Sterol loading rescue experiments were performed by incubation for 1 h at 37°C supplemented with 0.5% BSA, followed by removal and addition of 7.5% LPDS for imaging or subsequent analysis to limit prolonged exposure to MβCD. Comparison of the physical properties and phase separation behavior of sterols was previously described (Bacia et al., 2005 (link); Keller et al., 2004 (link); Megha et al., 2006 (link); Wenz and Barrantes, 2003 (link)).
7 dhc
Manufactured by Merck Group
Sourced in United States, United Kingdom
7-DHC is a biochemical compound used in research and development applications. It serves as a precursor for the synthesis of various substances and is commonly utilized in laboratory settings for experimental purposes.
Automatically generated - may contain errors
Lab products found in correlation
4β hydroxycholesterol, by Avanti Polar Lipids (2 mentions)
Methyl β cyclodextrin mβcd cholesterol complex, by Merck Group (2 mentions)
Acetonitrile, by Merck Group (2 mentions)
Cholesterol, by Merck Group (2 mentions)
Vitamin d3, by Merck Group (2 mentions)
Squalene, by Merck Group (1 mentions)
Db 5 fused silica capillary column, by Agilent Technologies (1 mentions)
Gcms qp2020, by Shimadzu (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
Agno3, by Merck Group (1 mentions)
Ferric ammonium citrate, by Merck Group (1 mentions)
Iron chloride hexahydrate, by Merck Group (1 mentions)
Hexanal, by Merck Group (1 mentions)
1 2 dimyristoyl sn glycero 3 phosphocholine, by Avanti Polar Lipids (1 mentions)
Ferric citrate, by Merck Group (1 mentions)
Pentanal, by Merck Group (1 mentions)
Deferoxamine mesylate salt, by Merck Group (1 mentions)
4 hydroxynonenal 4 hne, by Cayman Chemical (1 mentions)
Meoamvn, by Fujifilm (1 mentions)
Docosahexaenoicacid dha, by Nu-Chek Prep (1 mentions)
15 protocols using 7 dhc
1
Sterol-Methyl-β-Cyclodextrin Complex Formation
2
Sterol Extraction and GC-MS Analysis
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Sterol extraction was performed as previously described (Guo et al., 2018 ). Yeast cells were harvested by centrifugation at 12,000 rpm for 2 min after fermentation process and boiled in 3 N HCl for 5 min to break the cell wall. Cells were pelleted and washed by distilled water to remove the remaining HCl. After neutralizing by NaOH, saponification reaction of cells was carried out in 3 M NaOH-methanol solution at 60°C for 4 h. n-Hexane and silica sand were added for sterol extraction with vortex. The n-hexane phase was collected and dried by a centrifugal vacuum evaporator. N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA) was used to derivatize the obtained sterols (30°C for h) and samples were ready for GC-MS analysis.
Sterols were separated and analyzed by GCMS-QP2020 (SHIMADZU, Japan) using a DB-5 fused-silica capillary column (30 m × 0.25 mm i.d., film thickness 0.25 μm, J&W Scientific, CA). Mass spectra ranged at 50–800 m/z, and helium was used as the carrier gas. Operating conditions were inlet temperature 260°C, initial temperature 70°C for 2 min then ramp 30°C/min to 250°C then ramp 10°C/min to 280°C and held for 15 min. Finally, the temperature increased to 290°C at 5°C/min and was held for 5 min. Sterol standards (squalene, A1, A2, B3, B1, 7-DHC, etc.) were purchased from Sigma-Aldrich (United States).
Sterols were separated and analyzed by GCMS-QP2020 (SHIMADZU, Japan) using a DB-5 fused-silica capillary column (30 m × 0.25 mm i.d., film thickness 0.25 μm, J&W Scientific, CA). Mass spectra ranged at 50–800 m/z, and helium was used as the carrier gas. Operating conditions were inlet temperature 260°C, initial temperature 70°C for 2 min then ramp 30°C/min to 250°C then ramp 10°C/min to 280°C and held for 15 min. Finally, the temperature increased to 290°C at 5°C/min and was held for 5 min. Sterol standards (squalene, A1, A2, B3, B1, 7-DHC, etc.) were purchased from Sigma-Aldrich (United States).
3
MALDI-TOF Analysis of 7-Dehydrocholesterol
4
Evaluating Lipid Peroxidation Biomarkers
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7-DHC, ferric citrate, iron chloride hexahydrate,
ferric ammonium citrate, sulfate heptahydrate, a-tocopherol, deferoxamine
mesylate salt, pentanal, hexanal, 2-trans-heptenal,
2-trans-octenal, 2-trans-nonenal,
2,4-trans,trans-hexadienal, 2,4-trans,trans-nonadienal, and 2,4-trans,trans-decadienal were
obtained from Sigma-Aldrich. Retinol, 2,4-dinitrophenylhydrazine (DNPH),
and LpDNPH S10L cartridges were purchased from Thermo Fisher Scientific.
Retinal, coenzyme Q10, and vitamin D3 were purchased
from Chem-Impex Int’l Inc. Oleic acid (OA), linoleic acid (LA),
9,12,15-cis,cis,cis-octadecatrienoic acid (NLA 18:3),
arachidonic acid (AA), eicosapentaenoic acid (EPA), docosahexaenoic
acid (DHA), and 10,12-trans,cis-octadecadienoic acid
(CLA 18:2) were purchased from Nu-Chek Prep, Inc. 9,11,13-trans,trans,trans-octadecatrienoic acid (CLA 18:3) was obtained
from Sapphire North America. 1,2-dimyristoyl-sn-glycero-3-phosphocholine
(DMPC) was obtained from Avanti Polar Lipids. Imidazole ketone erastin
(IKE), RSL3, liproxstatin-1, and ferrostatin-1 were obtained from
MedChemExpress. 4-hydroxynonenal (4-HNE) was purchased from Cayman
Chemical. The radical initiator, MeOAMVN, was purchased from Wako
Chemicals.
ferric ammonium citrate, sulfate heptahydrate, a-tocopherol, deferoxamine
mesylate salt, pentanal, hexanal, 2-trans-heptenal,
2-trans-octenal, 2-trans-nonenal,
2,4-trans,trans-hexadienal, 2,4-trans,trans-nonadienal, and 2,4-trans,trans-decadienal were
obtained from Sigma-Aldrich. Retinol, 2,4-dinitrophenylhydrazine (DNPH),
and LpDNPH S10L cartridges were purchased from Thermo Fisher Scientific.
Retinal, coenzyme Q10, and vitamin D3 were purchased
from Chem-Impex Int’l Inc. Oleic acid (OA), linoleic acid (LA),
9,12,15-cis,cis,cis-octadecatrienoic acid (NLA 18:3),
arachidonic acid (AA), eicosapentaenoic acid (EPA), docosahexaenoic
acid (DHA), and 10,12-trans,cis-octadecadienoic acid
(CLA 18:2) were purchased from Nu-Chek Prep, Inc. 9,11,13-trans,trans,trans-octadecatrienoic acid (CLA 18:3) was obtained
from Sapphire North America. 1,2-dimyristoyl-sn-glycero-3-phosphocholine
(DMPC) was obtained from Avanti Polar Lipids. Imidazole ketone erastin
(IKE), RSL3, liproxstatin-1, and ferrostatin-1 were obtained from
MedChemExpress. 4-hydroxynonenal (4-HNE) was purchased from Cayman
Chemical. The radical initiator, MeOAMVN, was purchased from Wako
Chemicals.
5
Enzymatic Synthesis of Vitamin D Metabolites
6
Synthesis and Purification of Vitamin D Metabolites
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Lumisterol, 7DHC, D3 and 1,25(OH)2D3 were obtained from Sigma-Aldrich (St. Louis, MO). 20(OH)7DHC, 20(OH)L3, pL, 17(OH)pL and 17,20(OH)2pL were synthesized as previously described32 (link), 34 (link), 43 (link); while 22(OH)L3, 20,22(OH)2L3 and 24(OH)L3 were produced from L3 enzymatically using purified bovine CYP11A140 (link). These compounds were purified by reverse-phase HPLC; their structures and purities were determined by NMR and mass spectrometry32 (link), 34 (link), 40 (link), 43 (link).
7
Vitamin D and Sterol Analytical Methods
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All organic solvents and reagents used throughout this study for sample and mobile phase preparation were analytical grade. The organic solvents were pentane (RCI LABSCAN, HPLC grade), ethyl ether (LOBA CHEMIE), acetonitrile (HPLC grade), methanol (both of LC-MS and HPLC grade), and sodium L-ascorbate (crystalline > 98%, Sigma-Aldrich Corp., St. Louis, MO, USA). The reagents were potassium hydroxide (45%) and sodium chloride (10%, Ajax Finechem, Taren Point, Australia).
Standards were purchased at the highest purity (>95%) from suppliers, including vitamin D3 (product No. 47763), vitamin D2 (Suplelco, Bellefonte, PA, USA, product No. 47768), tri-deuterated vitamin D2 used as an internal standard (2H3-D2) (Sigma-Aldrich Corp., St. Louis, MO, USA, product No. V-026-1ML), ergosterol (ThermoFisher, Winsford, UK, product No. B23840), 7-DHC (Sigma-Aldrich-Corp., St. Louis, MO, USA, product No. 30800), 25-OH D2 (Enzo, New York, NY, USA, product No. BML-DM101), and 25-OH D3 (Enzo., New York, NY, USA, product No. BML-DM100).
Standards were purchased at the highest purity (>95%) from suppliers, including vitamin D3 (product No. 47763), vitamin D2 (Suplelco, Bellefonte, PA, USA, product No. 47768), tri-deuterated vitamin D2 used as an internal standard (2H3-D2) (Sigma-Aldrich Corp., St. Louis, MO, USA, product No. V-026-1ML), ergosterol (ThermoFisher, Winsford, UK, product No. B23840), 7-DHC (Sigma-Aldrich-Corp., St. Louis, MO, USA, product No. 30800), 25-OH D2 (Enzo, New York, NY, USA, product No. BML-DM101), and 25-OH D3 (Enzo., New York, NY, USA, product No. BML-DM100).
8
Sterol-Methyl-β-Cyclodextrin Complex Formation
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Methyl-β-cyclodextrin (MβCD)-cholesterol complex commercially available (Sigma, C4951). For MβCD-sterol complexation, 50 mg/mL sterol stocks in 1:1 chloroform:methanol were prepared for desmosterol, lathosterol, cholestenone, cholesterol sulfate, 4β-hydroxycholesterol (Avanti Polar Lipids), 7DHC and cholesterol (Sigma or Avanti Polar Lipids). Stoichiometry of MβCD-cholesterol complexes typically consists of one cholesterol molecule entrapped within two CDs (1:2 molar ratio). We found optimal loading conditions using a 1:7 molar ratio, where desired sterol concentration was dried under nitrogen flow, followed by addition of 5 mM MβCD (Alfa Aesar, 1303.31 g/mol) prepared in serum-free DMEM. Sterol suspensions were sonicated for 5 min and incubated at 37°C with agitation overnight. Sterol crystals were subsequently removed via 0.45 μm filtration. Sterol loading rescue experiments were performed by incubation for 1 h at 37°C supplemented with 0.5% BSA, followed by removal and addition of 7.5% LPDS for imaging or subsequent analysis to limit prolonged exposure to MβCD. Comparison of the physical properties and phase separation behavior of sterols was previously described (Bacia et al., 2005 (link); Keller et al., 2004 (link); Megha et al., 2006 (link); Wenz and Barrantes, 2003 (link)).
9
Synthesis and Characterization of Vitamin D Metabolites
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7DHC, vitamin D3, 25(OH)D3, and 1,25(OH)2D3 were obtained from Sigma-Aldrich (St. Louis, MO). 7DHP was synthesized as described previously38 (link)53 (link), while 22(OH)7DHC and 20,22(OH)27DHC 20(OH)D3 were produced from 7DHC enzymatically using purified bovine CYP11A149 (link). 20(OH)D3, 22(OH)D3, 20,22(OH)2D3, 20,23(OH)2D3 and 17,20,23(OH)3D3 were produced from vitamin D3 using purified CYP11A1, while 1,20(OH)2D3 was similarly produced from 1α(OH)D323 (link)25 (link)57 (link)60 (link). 20,24(OH)2D3, 20,25(OH)2D3 and 20,26(OH)3D3, were produced from 20(OH)D3 using recombinant CYP27A1 or CYP24A128 (link)29 (link). 1,20,24(OH)3D3, 1,20,25(OH)3D3 and 1,20,26(OH)3D3 were made by by 1α-hydroxylation of either 20,24(OH)2D3, 20,25(OH)2D3 or 20,26(OH)2D3 using recombinant CYP27B132 (link). These compounds were purified by reverse-phase HPLC, and their structures and purities were determined by NMR and mass spectrometry23 (link)25 (link)28 (link)29 (link)32 (link)36 (link)48 (link)49 (link)57 (link)60 (link).
10
Transcription and Translation Inhibition Assay
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The ECIS system was used with 8-well plates (8W10E+, Applied Biophysics), with cells seeded at 2.5x104 cells/well. Transcription and translation inhibition experiments were performed using a Roche SP Xcelligence transendothelial system and 96-well assay plates with cells seeded at 1x104 cell/well. Treatment in all assays, unless otherwise denoted were 10μm D3 (Tocris), 7-DHC (Sigma) or vehicle (0.5% DMSO) and simultaneous cytokine addition of either TNF-α (2ng/mL) or IL-1β (10ng/mL).
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