Per sample, 20 μg of proteins were loaded on a 12% sodium dodecyl sulfate-polyacrylamide gel (Bio-Rad), the electrophoresis subsequently ran for ten minutes at 50 V followed by four minutes at 180 V. Protein bands were stained with Coomassie blue (Sigma-Aldrich) and collected from the gel, followed by an in-gel digestion with a MassPREP robot (Waters, UK). First, the destaining of Coomassie blue was performed using 50% acetonitrile (Biosolve B.V., Netherlands) in 50 mM ABC buffer. Second, cysteines were reduced using 10 mM dithiothreitol (Sigma-Aldrich) in 50 mM ABC buffer, followed by alkylation for 20 minutes using 55 mM iodoacetamide (Sigma-Aldrich) in 100 mM ABC buffer. This step was carried out in the dark to avoid UV degradation of the iodoacetamide. The samples were subsequently washed with 50 mM ABC buffer and dehydrated using acetonitrile. The dehydrated proteins were digested using 6 ng/μl trypsin (Promega, Netherlands) in 50 mM ammonium bicarbonate (ABC) buffer. The solution was incubated at 37°C for five hours. Lastly, the peptides were extracted from the gel using 3 × 50 μl of 2% acetonitrile and 1% formic acid (Biosolve B.V.). The samples were concentrated to 50 μl using a speedvac vacuum concentrator (Eppendorf SE) (Figure 1 ).
Acetonitrile
Manufactured by Biosolve
Sourced in France, Netherlands, Germany, United States, Belgium, Switzerland
Acetonitrile is a colorless, volatile liquid commonly used as a solvent in various laboratory applications. It has a low boiling point and is miscible with water and many organic solvents. Acetonitrile is a versatile chemical that can be used in various analytical and chemical processes.
Automatically generated - may contain errors
Lab products found in correlation
Methanol, by Biosolve (71 mentions)
Formic acid, by Biosolve (52 mentions)
Formic acid, by Merck Group (30 mentions)
Ammonium acetate, by Merck Group (14 mentions)
Ammonium formate, by Merck Group (13 mentions)
Sodium hydroxide (naoh), by Merck Group (12 mentions)
Acetone, by Biosolve (10 mentions)
Isopropanol, by Biosolve (10 mentions)
Dithiothreitol (dtt), by Merck Group (9 mentions)
Hydrochloric acid (hcl), by Merck Group (9 mentions)
Acetic acid, by Merck Group (8 mentions)
Ammonium formate, by Biosolve (7 mentions)
Methanol, by Merck Group (7 mentions)
Piperidine, by Biosolve (6 mentions)
Trifluoroacetic acid, by Biosolve (6 mentions)
Ammonium bicarbonate, by Merck Group (6 mentions)
Lc ms grade water, by Biosolve (6 mentions)
Ammonium hydroxide, by Merck Group (6 mentions)
Catechin, by Merck Group (6 mentions)
Trifluoroacetic acid, by Merck Group (6 mentions)
171 protocols using acetonitrile
1
In-Gel Protein Digestion Protocol
2
Synthesis and Characterization of Gold Nanoparticles for Immunological Studies
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All chemicals were purchased from Sigma-Aldrich unless stated otherwise. CTAB (≥99%, H6269) and HAuCl4 × 3H2O (49% Au basis, G4022) were purchased from Sigma-Aldrich. Silver nitrate (99.999%) and Oxyma Pure were purchased from Carl Roth GmbH. TFA, piperidine, DMF, DCM, methanol, and acetonitrile were purchased from Biosolve. TEM grids (Formvar/Carbon, 200 mesh, on copper support) were purchased from Electron Microscopy Sciences. FCS, penicillin/streptomycin mixture, l -glutamate, DMEM and RPMI 1640 cell culture media were purchased from Lonza. Mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) was purchased from PeproTech. CD4+ and CD8+ isolation kits (via MACS magnetic separation) were purchased from Miltenyi Biotech. LDH leakage assay (LDH-Cytox™ Assay Kit) and the specific antibody against the whole MHC-I/OVA257–264 complex (PE anti-mouse H-2Kb bound to OVA257–264 antibody, Kb-OVA257–264) were purchased from Biolegend. ELISA standards (IL-12 and IL-1β) and antibodies, TMB substrate, as well as immunostaining antibodies were purchased from eBioscience.
3
Synthesis of α-Ribazole from Sigma-Aldrich
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Standard compounds were purchased from Sigma-Aldrich (Steinheim, Germany) in analytical purity. α-Ribazole was synthesized according to Wienhausen et al. [25 (link)]. Ultrapure water was obtained by filtration of deionized water in an arium® pro ultrapure water system (Sartorius, Göttingen, Germany). Acetonitrile and methanol were purchased from Biosolve (Dieuze, France) in ULC/MS grade.
4
Alpha-2-HS Glycoprotein Purification and Analysis
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hFet (alpha-2-HS glycoprotein;
Uniprot Code: P02765), bFet (bovine fetuin; Uniprot Code: P12763), and rhFet (recombinant alpha-2-HS
glycoprotein expressed in HEK293 cells), dithiothreitol (DTT), iodoacetamide
(IAA), trifluoroacetic acid (TFA), ammonium bicarbonate (ABC), and
ammonium acetate (AMAC) were purchased from Sigma-Aldrich (Steinheim,
Germany). Acetonitrile was purchased from Biosolve (Valkenswaard,
The Netherlands). Sequencing-grade trypsin was obtained from Promega
(Madison, WI). Gluc-C, Lys-C, PNGaseF,32 (link) and Sialidase were obtained from Roche (Indianapolis, IN). Alkaline
phosphatase was purchased from New England Biolabs (Ipswich, MA).
Uniprot Code: P02765), bFet (bovine fetuin; Uniprot Code: P12763), and rhFet (recombinant alpha-2-HS
glycoprotein expressed in HEK293 cells), dithiothreitol (DTT), iodoacetamide
(IAA), trifluoroacetic acid (TFA), ammonium bicarbonate (ABC), and
ammonium acetate (AMAC) were purchased from Sigma-Aldrich (Steinheim,
Germany). Acetonitrile was purchased from Biosolve (Valkenswaard,
The Netherlands). Sequencing-grade trypsin was obtained from Promega
(Madison, WI). Gluc-C, Lys-C, PNGaseF,32 (link) and Sialidase were obtained from Roche (Indianapolis, IN). Alkaline
phosphatase was purchased from New England Biolabs (Ipswich, MA).
5
Mass Spectrometry Sample Preparation
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All chemicals were of the highest obtainable purity. Water, formic acid (FA), trifluoroacetic acid (TFA), acetonitrile (ACN), and methanol (MeOH) were purchased from Biosolve B.V. (Valkenswaard, the Netherlands), alpha-cyano-4-hydroxycinnamic acid (HCCA), 2.5-dihydroxybenzoïc (2,5-DHB), sinapinic acid (SA), lysozyme C and cytochrome C, ammonium bicarbonate (AB), dithiothreitol (DTT) and iodoacetamide (IAA) from Sigma-Aldrich (Saint-Quentin Fallavier, France), bradykinin (BK) from PolyPeptide Group (Strasbourg, France), Phosphatidyl Choline 15:0/15:0 lipid species (PC) from Avanti Polar Lipid (Alabaster, Alabama, USA), carmine from George T. GURR LTD (London, England) and Congo red from Microcolor (Boulogne, Seine, France). Trypsin (sequencing grade modified trypsin, porcine) was purchased from Promega (Charbonnieres, France).
6
Purification and Tryptic Digestion of CD8+ T Cells
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Cell pellets of FACS‐purified CD8+ T cells were washed in ice cold PBS, snap frozen in liquid nitrogen and stored at ‐80°C until further processing. Tryptic peptides were prepared as previously reported with minor adjustments (Kulak et al., 2014). In brief, cells were lysed in 1% (v/v) Sodium Deoxy Cholate (Sigma), 10 mM TCEP (Thermo Scientific), 40 mM ChloroAcetamide (Sigma Aldrich), 100 mM TRIS‐HCl pH 8.0 (Life Technologies) and HALT protease/phopsphatase inhibitor cocktail (Thermo Scientific). Samples were incubated at 95°C for 5 minutes and sequentially sonicated for 10 minutes in a Bioruptor Pico (Diagenode). After the addition of 50 mM ammonium bicarbonate (Sigma) containing 6.25 μg/mL Trypsin Gold (Promega), the samples were digested overnight at room temperature. After digestion, the samples were acidified by the addition of trifluoroacetic acid (Thermo Scientific) and loaded on an in‐house prepared SDB‐RPS STAGEtips (Empore). The peptides were desalted and eluted in 5% (v/v) ammonium hydroxide (Sigma) and 80% (v/v) acetonitrile (BioSolve). The sample volume was reduced by SpeedVac and supplemented up to 10 μl with 2% (v/v) acetonitrile and 0.1% (v/v) TFA. Peptide concentrations were determined using a colorimetric peptide kit (Thermo Scientific) and 3 μl of each sample (containing 0.75 μg peptides) was injected for MS data acquisition.
7
Nanoscale Liquid Chromatography-Tandem Mass Spectrometry
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The 18 fractions were analyzed on a nanoAcquity UPLC system (Waters) directly connected to an LTQ Orbitrap Velos Pro instrument (Thermo Scientific) via a Proxeon nanospray source. The peptides were first trapped on a nanoAcquity Symmetry C18 5 µm 180 µm×20 mm (Waters) trapping column and further separated on a nanoAcquity BEH C18 1.7 µm 75 µm×200 mm (Waters) analytical column. The mobile phases were 0.1% formic acid in water and 0.1% formic acid in acetonitrile (Biosolve). The applied three-step gradient was 120-min long, ranging from 3% to 40% acetonitrile. The flow rate was 300 nl/min. The eluent was directly introduced into the mass spectrometer through a Pico-Tip emitter 360-µm outer diameter×20-µm inner diameter, 10-µm tip (New Objective). The capillary temperature was set to 300°C, and the applied spray voltage was 2.2 kV. The mass range of the full scan MS spectra was 300–1700 m/z. MS1 spectra were recorded in profile mode in the Orbitrap. The resolution was set to 30,000. Lock mass correction was used for internal calibration using a background ion at m/z 445.12003. The 15 most abundant parent ions were subjected to fragmentation by using collision-induced dissociation. The normalized collision energy was set to 40. Charge state screening was enabled with only multiply-charged ions being selected for fragmentation.
8
Synthesis and Characterization of PLGA Nanoparticles
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Glycolide, stannous 2-ethyl hexanoate (SnOct 2 ), 2,2′-azobis(2methaylpropionitrile) (AIBN), 4,4-azobis(4-cyanopentanoic acid) (ABCPA), hydroxypropyl methacrylate (HEMA), formic acid, 1,1′-carbonyldiimidazole (CDI), 4-(N, N-dimethylamino) pyridine (DMAP), mineral oil (M8410), deuterated dimethyl sulphoxide (DMSO-d 6 ), perchloric acid (HClO 4 ), and blue dextran (molecular weight 2 000 000 Da) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Dichloromethane (DCM), hexane, ethyl acetate, methanol, acetonitrile, dimethyl sulphoxide (DMSO, dried over 3 Å molecular sieves), and acetone were purchased from Biosolve (Valkenswaard, The Netherlands). Irgacure 2959 was obtained from Ciba Specialty Chemicals Inc. (Hercules, USA). ABIL EM 90 was provided from Evonik Industries AG (Essen, Germany). HPMAm and HEMAm were synthesized as described previously. [34] [35] [36] poly(DL-lactic-co-glycolic acid) (PLGA, PDLG 5004A, Purasorb) was obtained from Corbion, Gorichem, the Netherlands. PLGA nanoparticles with a size of 295 nm as measured by dynamic light scattering (DLS) were prepared using a solvent evaporation method. [ 37 ] All other chemicals and solvents were used as received.
9
Extraction and Characterization of Coccoloba cowellii
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Acros Organics (Geel, Belgium) and Fisher Scientific (Leicestershire, UK) were the companies from which all analytical grade solvents, such as n-hexane, chloroform, dichloromethane, ethyl acetate, isopropyl alcohol, n-butanol, methanol, and dimethyl sulfoxide (DMSO), were purchased. HPLC solvents, such as methanol and acetonitrile, were purchased from Fisher Scientific, while UPLC-grade solvents, such as acetonitrile and formic acid, were purchased from Biosolve (Valkenswaard, the Netherlands). Finally, methanol-d4 (≥99.8% D) was obtained from Sigma-Aldrich (St. Louis, MO, USA); Milli-Q quality water was dispensed using a Milli-Q system from Millipore (Bedford, MA, USA) and subsequent membrane filtration at 0.22 μm was performed before use. Coccoloba cowellii leaves were collected near Albaisa in the municipality of Camagüey, Cuba (Lat. 21.43615, Long. −77.83253), and were taxonomically identified by the curator of the herbarium “Julián AcuñaGalé” of the University of Camagüey (HIPC, http://sweetgum.nybg.org/science/ih/herbarium-details/?irn=124935 (accessed on 20 August 2021)), where a sample specimen (number 12057) was deposited.
10
Quantification of Cortisone and Cortisol
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Cortisone and cortisol standards were purchased from Cerilliant (Round Rock, USA), item numbers C-130 and C-106. 13 C 3 -Cortisol and 13 C 3 -Cortisone were obtained from IsoSciences (Ambler, USA), item numbers S14466-0.1 and S14465-0.1. LC-MS grade methanol (catalogue number (CN):13684101), acetonitrile (CN:12401), formic acid (CN:0006914131BS) were purchased from Biosolve (Valkenswaard, The Netherlands). Ammonium fluoride were obtained from Sigma-Aldrich (CN: 338869-25G) (St Louis, USA), ammonium hydroxide from Merck (CN: 5.33003.0050) (Darmstadt, Germany). Ultra-pure water was produced in house by Q-POD Ultrapure Remote Dispenser from Merck Millipore (Burlington, USA).
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