To obtain vaccine compositions, ovalbumin (Sigma-Aldrich, USA) (10 μg/dose) was mixed with individual CLR agonists: TDB, curdlan, and furfurman (all, InvivoGen, USA) (50 μg/dose) in 100 μL sterile PBS. Interactions between antigen and individual CLR agonists were measured by surface plasmon resonance imaging. Squalene-based vaccine formulations were made by thorough mixing ovalbumin antigen (10 μg/dose) and CLR agonists (50 μg/dose) with 10 μL Squalene (Sigma-Aldrich, USA) and 15 μL of 10% Tween 80 (PanReac AppliChem, Germany). The volume of each formulation was adjusted with PBS to a total of 200 μL/dose. Mixtures were further sonicated (A—20%, 30 sec) (Branson, USA). The emulsions were stable for two weeks (the particle size was about 350 nm and did not change). Stability was controlled in the Zetasizer Nano ZS (Malvern, UK). Mice were immunized by the subcutaneous route (s.c., volume of 100 μL) at the base of the tail. Two immunizations were given with 2 weeks interval.
Squalene
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom, France, China, Spain, Japan, Italy
Squalene is a natural organic compound that is widely used in laboratory settings as a component in various types of lab equipment. It functions as a key ingredient in the formulation of various substances used in scientific research and testing. Squalene is derived from natural sources and is a commonly used material in the manufacture of specialized laboratory equipment and consumables.
Automatically generated - may contain errors
Lab products found in correlation
Cholesterol, by Merck Group (15 mentions)
α tocopherol, by Merck Group (13 mentions)
Tween 80, by Merck Group (13 mentions)
Stigmasterol, by Merck Group (11 mentions)
Campesterol, by Merck Group (11 mentions)
β sitosterol, by Merck Group (11 mentions)
Methanol, by Merck Group (9 mentions)
Stearic acid, by Merck Group (9 mentions)
Palmitic acid, by Merck Group (8 mentions)
Lanosterol, by Merck Group (7 mentions)
Gallic acid, by Merck Group (7 mentions)
Hexane, by Merck Group (7 mentions)
Ergosterol, by Merck Group (6 mentions)
Simvastatin, by Merck Group (6 mentions)
Oleic acid, by Merck Group (6 mentions)
Acetonitrile, by Merck Group (6 mentions)
Chloroform, by Merck Group (6 mentions)
Mevalonolactone, by Merck Group (5 mentions)
2 2 diphenyl 1 picrylhydrazyl (dpph), by Merck Group (5 mentions)
5α cholestane, by Merck Group (5 mentions)
153 protocols using squalene
1
Optimizing Vaccine Formulations with CLR Agonists
2
Immunization and Antibody Quantification
3
Vaccine Formulation and Immunization in Mice
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Young (6–8 weeks old) and aged (18–22 months old) wild-type C57BL/6 female mice were purchased from Harlan, UK. All animal procedures were performed according to UK Home Office and institutional regulations.
CASAC vaccine comprised of an oil-in-water emulsion consisting of Tween-80 and squalene (all Sigma, UK), as previously described [14 (link)]. The tween/squalene mixture was sonicated and mixed at a 1:1 ratio with PBS containing: 50 μg polyI:C (TLR3 agonist; Sigma), 25 μg CpG 1826 (TLR9 agonist; Eurofins, UK), 100 ng mouse recombinant IFN-γ (Peprotech, UK), 100 μg ISQAVHAAHAEINEAGR (ovalbumin (OVA)-derived MHC-class II (H-2IAb)-restricted peptide) and 100 μg SIINFEKL (SIL; OVA-derived MHC-class I (H-2Kb)-restricted peptide) or SVYDFFVWL (SVL; tyrosinase related protein (TRP)-2-derived MHC-class I (H-2Kb)-restricted peptide; all PPR, UK). Alternatively, 100 μg of SIL or SVL was emulsified with Complete Freund’s Adjuvant (CFA) for the first vaccination, and Incomplete FA (IFA; all Sigma) for subsequent vaccinations at a 1:1 (vol/vol) ratio. All vaccine formulations were administered intradermally on days 0, 10, 20 and 30 (±1 day) in 100 μL final volume (50 μL/flank).
CASAC vaccine comprised of an oil-in-water emulsion consisting of Tween-80 and squalene (all Sigma, UK), as previously described [14 (link)]. The tween/squalene mixture was sonicated and mixed at a 1:1 ratio with PBS containing: 50 μg polyI:C (TLR3 agonist; Sigma), 25 μg CpG 1826 (TLR9 agonist; Eurofins, UK), 100 ng mouse recombinant IFN-γ (Peprotech, UK), 100 μg ISQAVHAAHAEINEAGR (ovalbumin (OVA)-derived MHC-class II (H-2IAb)-restricted peptide) and 100 μg SIINFEKL (SIL; OVA-derived MHC-class I (H-2Kb)-restricted peptide) or SVYDFFVWL (SVL; tyrosinase related protein (TRP)-2-derived MHC-class I (H-2Kb)-restricted peptide; all PPR, UK). Alternatively, 100 μg of SIL or SVL was emulsified with Complete Freund’s Adjuvant (CFA) for the first vaccination, and Incomplete FA (IFA; all Sigma) for subsequent vaccinations at a 1:1 (vol/vol) ratio. All vaccine formulations were administered intradermally on days 0, 10, 20 and 30 (±1 day) in 100 μL final volume (50 μL/flank).
4
Dietary Squalene Intervention in Rabbits
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
During 4 weeks, 12 male New Zealand White rabbits (1.2 kg body weight) were fed with a regular diet enriched with 1% of sunflower oil for the control group (n=6), and with 1% of sunflower oil and 0.5% of squalene (Sigma-Merck, Darmstadt, Germany) for the squalene group (n=6). Sunflower oil was used to dissolve squalene and 1% was empirically found to be perfectly embedded into diet pellets without requiring milling and pelleting again. Regular diet pellets were sprayed with sunflower oil or squalenecontaining sunflower oil. Diets were prepared weekly and changed every two days to reduce squalene oxidation. Intake and body weights were monitored every 2 days. At the end of the 4-week dietary intervention, food was withdrawn for 18 hours, and the rabbits were weighed and then sacrificed. This fasting time was selected according to studies carried in these animals using dietary fats 38, 39 . Venous blood samples, the livers and the jejunums were obtained for assays. The samples were immediately frozen in liquid nitrogen and stored at -80 ºC. Animals were handled and killed observing guidelines from the European Union for care and use of laboratory animals in research (Directive 2010/63/UE), and the protocols were approved by the Ethics Committee for Animal Research of the University of Zaragoza (PI47/10).
5
Molecular cloning and yeast manipulation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Potassium hydroxide pellets, acetone (≥98%), ergosterol (≥98%) and squalene (≥98%) used as standards and petroleum ether (b.p. = 40–60°C) were all purchased from Sigma-Aldrich, while ethanol absolute was supplied from Scharlau.; Sclareol, kindly provided by VIORYL, SA, (−)-trans-caryophyllene (Sigma, C9653-5), were used as standard compounds; MyTaq DNA polymerase (BIO-21105, Bioline), and Accuzyme DNA polymerase (BIO-21051, Bioline) were used in PCR amplifications; NucleoSpin Plasmid Kit (REF 740588.250, Macherey-Nagel) was used for plasmid DNA purification; QIAquick Gel Extraction Kit (#28704, Qiagen) was used for gel extraction and DNA purification.
Yeast media: D (+)-Glucose monohydrate (16301, Sigma); Yeast Nitrogen Base w/o AA, carbohydrate & w/AS (Y2025, US Biologicals); Complete Minimal (CM) medium is composed of 0.13% (w/v) dropout powder (all essential amino acids) [45 ], 0.67% (w/v) yeast nitrogen base w/o AA, 2% glucose; TOPO TA Cloning Kit Dual Promoter (K4610-20, Invitrogen). All yeast transformations were done by lithium acetate transformation.
Yeast media: D (+)-Glucose monohydrate (16301, Sigma); Yeast Nitrogen Base w/o AA, carbohydrate & w/AS (Y2025, US Biologicals); Complete Minimal (CM) medium is composed of 0.13% (w/v) dropout powder (all essential amino acids) [45 ], 0.67% (w/v) yeast nitrogen base w/o AA, 2% glucose; TOPO TA Cloning Kit Dual Promoter (K4610-20, Invitrogen). All yeast transformations were done by lithium acetate transformation.
6
Macrophage Polarization and Phagocytosis
7
Squalene Dietary Effects on Rabbits
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The experimental animals used were 10 male wild-type New Zealand white rabbits obtained from Servicio de Apoyo a la Investigación Animal (Universidad de Zaragoza). Animals were divided into two groups, the first fed a control diet enriched with 1% of sunflower oil (n = 5) and the second fed a diet containing 1% of sunflower oil and 0.5% of squalene (Sigma-Merck, Darmstadt, Germany) (n = 5). Initial body weight for control and experimental groups was 1560 ± 201 and 1410 ± 203 g, respectively. Taking into account consumed food and body weight, the dose was equivalent to 0.6 mg/kg/day [20 (link)]. Fresh diets were prepared weekly and changed each 2 days to reduce oxidation of squalene. The animals were fed the experimental diets for 4 weeks and they were well tolerated.
Body mass and dietary intake were recorded every 2 days. After dietary intervention, rabbits were starved for 18 h, then weighed and euthanized by cervical dislocation, and livers were obtained. An aliquot of each sample was stored in neutral formaldehyde and the remaining was frozen immediately in liquid nitrogen.
Body mass and dietary intake were recorded every 2 days. After dietary intervention, rabbits were starved for 18 h, then weighed and euthanized by cervical dislocation, and livers were obtained. An aliquot of each sample was stored in neutral formaldehyde and the remaining was frozen immediately in liquid nitrogen.
8
UV-Induced Chemical Mixture Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All the chemicals involved such as squalene, benzoyl peroxide, AIBN, oleic, linoleic and arachidonic acids were purchased from Sigma-Aldrich with exception of DMSO-d6 purchased from FluoroChem and were used without further purification. DMSO-d6 was used to record all the 1H and DOSY NMR of the mixtures obtained after UV exposure. The microscope slides used to deposit the sample were purchased from Sigma-Aldrich.
Single-use 5 mm NMR tubes (Product code 502-7) were purchased from GPE Scientific.
Single-use 5 mm NMR tubes (Product code 502-7) were purchased from GPE Scientific.
9
Sterol Extraction and GC-MS Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
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).
10
Synthesis of PEGylated Squalene Nanocarriers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Squalene (SQ) (purchased from Sigma, ≥98%), 1,3,5-benzenetrialdehyde (TA) (purchased from Manchester Organics, 98%), poly-(ethyleneglycol)-bis(3-aminopropyl) terminated (NH2-PEG-NH2) (Mn~1500 g/mol) (purchased from Aldrich, Slovakia), and branched polyethylenimine (2000 Da, 50 wt % in H2O) (PEI2000) (purchased from Aldrich, St. Louis, MO, USA). All other chemicals were purchased from Sigma-Aldrich Chemie GmbH (Steinheim, Germany) and used without further purification. PEGylated Squalene (SQ-PEG-NH2) was prepared as previously described [34 (link),37 ]. All reagents and solvents were purchased from commercial sources.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!