For B/T cells co-cultures, naïve or memory B cells were co-cultured with CD4+ T cells (2 × 104 B cells - 2 × 104 CD4 T cells) in the presence of endotoxin-reduced SEB (500 ng/ml; Sigma Aldrich) in cRPMI supplemented with 10% heat-inactivated FBS. Where described, anti-IL10 (10 μg/ml - clone JES3–9D7 - Biolegend LEAF™ purified antibody), anti-SUCNR1/GPR91 (20 μg/ml – Novus Biological), anti- IL21R (10 μg/ml - clone 17A12 - Biolegend LEAF™ purified antibody) or succinate (2 mM – Sigma-Aldrich) were added during the co-cultures. Sodium azide and other preservatives were removed from the antibody preparations by protein desalting with Zeba Spin Desalting Columns (7K MWCO; Thermo Fisher). IgM and IgG concentrations were measured at day 12 in the corresponding supernatants with Flex Set Kit (BD Biosciences). For T cell-independent B cell differentiation, naïve B cells (5 × 104 cells) were co-cultured with irradiated (77Gy) human-CD40L transfected fibroblasts43 (link) (0.5 × 104 cells) in cRPMI supplemented with 10% heat-inactivated FBS. Recombinant human IL10 (Biolegend, 500 ng/ml) and/or succinate (2 mM – Sigma-Aldrich) were added during the co-cultures. IgG and IgM concentrations were measured at day 12 in the corresponding supernatants with Flex Set Kit (BD Biosciences).
Succinate
Manufactured by Merck Group
Sourced in United States, Germany, Japan, United Kingdom, Macao, France, Italy
Succinate is a laboratory equipment product that serves as a chemical compound. It functions as a dicarboxylic acid and is a key intermediate in the citric acid cycle, a central metabolic pathway in many organisms.
Automatically generated - may contain errors
Lab products found in correlation
Rotenone, by Merck Group (60 mentions)
Malate, by Merck Group (52 mentions)
Glutamate, by Merck Group (41 mentions)
Antimycin a, by Merck Group (39 mentions)
Adenosine diphosphate (adp), by Merck Group (35 mentions)
Oligomycin, by Merck Group (29 mentions)
Pyruvate, by Merck Group (28 mentions)
Ascorbate, by Merck Group (19 mentions)
Bovine serum albumin (bsa), by Merck Group (16 mentions)
Digitonin, by Merck Group (15 mentions)
Sucrose, by Merck Group (15 mentions)
Kh2po4, by Merck Group (12 mentions)
Ethylene glycol tetraacetic acid (egta), by Merck Group (12 mentions)
Mannitol, by Merck Group (12 mentions)
Malonate, by Merck Group (12 mentions)
Taurine, by Merck Group (11 mentions)
Hepes, by Merck Group (11 mentions)
Adenosine triphosphate (atp), by Merck Group (11 mentions)
Cytochrome c, by Merck Group (10 mentions)
Dithiothreitol (dtt), by Merck Group (8 mentions)
153 protocols using succinate
1
B/T Cell Co-culture Assay for Immunoglobulin Production
2
Mitochondrial Bioenergetics Assay
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Mannitol, sucrose, bovine serum albumin (BSA), N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) potassium salt, potassium phosphate monobasic anhydrous (KH2PO4), magnesium chloride (MgCl2), ethylene-diamine-tetra-acetic acid (EDTA), ethylene-glycol-tetra-acetic acid (EGTA), pyruvate, malate, glutamate, succinate, β-hydroxybutyrate, α-ketoglutarate, adenosine-5′-diphosphate (ADP), oligomycin A, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), rotenone, succinate, dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO, USA). BCA protein assay kit was purchased from Pierce (Rockford, IL). Both NAD(t) and FAD(t) content assessments kits were purchased from Sigma-Aldrich (St. Louis, MO, USA). Mitochondrial substrates and inhibitors stock solutions were prepared and aliquots stored at −80°C.
3
B/T Cell Co-culture Assay for Immunoglobulin Production
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For B/T cells co-cultures, naïve or memory B cells were co-cultured with CD4+ T cells (2 × 104 B cells - 2 × 104 CD4 T cells) in the presence of endotoxin-reduced SEB (500 ng/ml; Sigma Aldrich) in cRPMI supplemented with 10% heat-inactivated FBS. Where described, anti-IL10 (10 μg/ml - clone JES3–9D7 - Biolegend LEAF™ purified antibody), anti-SUCNR1/GPR91 (20 μg/ml – Novus Biological), anti- IL21R (10 μg/ml - clone 17A12 - Biolegend LEAF™ purified antibody) or succinate (2 mM – Sigma-Aldrich) were added during the co-cultures. Sodium azide and other preservatives were removed from the antibody preparations by protein desalting with Zeba Spin Desalting Columns (7K MWCO; Thermo Fisher). IgM and IgG concentrations were measured at day 12 in the corresponding supernatants with Flex Set Kit (BD Biosciences). For T cell-independent B cell differentiation, naïve B cells (5 × 104 cells) were co-cultured with irradiated (77Gy) human-CD40L transfected fibroblasts43 (link) (0.5 × 104 cells) in cRPMI supplemented with 10% heat-inactivated FBS. Recombinant human IL10 (Biolegend, 500 ng/ml) and/or succinate (2 mM – Sigma-Aldrich) were added during the co-cultures. IgG and IgM concentrations were measured at day 12 in the corresponding supernatants with Flex Set Kit (BD Biosciences).
4
Mitochondrial Respiration Profiling
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Tissue respiration was performed using an O2K high-resolution respirometer (Oroboros, Austria). Freshly isolated tissues were rapidly weighed (BAT 2–5 mg), minced, and homogenized using a PBI-shredder PBI set (Oroboros, Austria). Homogenized tissue was further re-suspended in 2 mL of MiR05 medium (0.5 mM of EGTA, 3 mM of MgCl2, 60 mM of K-lactobionate, 20 mM of Taurine, 10 mM of KH2PO4, 20 mM of HEPES, 110 mM of Sucrose, and 1 g/L of BSA (essentially fatty acid free)). Respiratory oxygen flux was measured in real time and expressed as picomoles of O2 per second per mg of tissue. 5 mM of pyruvate (Sigma), 2 mM of malate (Sigma), and 1 mM of ADP (Sigma) were added to stimulate complex I respiration. 1 µM of rotenone (Sigma) and 5 mM of succinate (Sigma) were added to test complex II respiration. 2 mM of ascorbate and 0.5 mM of N, N, N, N-tetramethyl-p-phenylenediamine (TMPD) (Sigma) were used to test complex IV respiration. To measure uncoupled respiration, succinate (Sigma) was used as the substrate, and 1 μM of oligomycin (Sigma) was added to determine the coupled respiration. Uncoupled respiration was calculated using the respiration rate after oligomycin deducted by respiration rate after antimycin A (1 μM) (Sigma) inhibition. To measure mitochondrial β-oxidation, palmitoyl-carnitine (5 μM) (Sigma) was added to the chamber.
5
Mitochondrial Dysfunction Evaluation Protocol
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Apocynin, apyrase, bovin serum albumin, butylated hydroxytoluene, cytochrome C, dichlorofluorescin diacetate (DCFH-DA), diphenyleneiodonium (DPI), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), dithiotreitol (DTT), L-lactic dehydrogenase (EC 1.1.1.27), leupeptin, NAD+, NADH, PGE1, piceatannol, phenylmethylsulfonyl fluoride (PMSF), PP2 analogue (PP2), protease inhibitor cocktail (Cat. N° P8340), superoxide dismutase (SOD), thiobarbituric acid (TBA), digitonin, pyruvate, malate, succinate, ouabain, ampicillin, di-adenosine-5'penta-phosPHAte, rotenone, antimycin A, ADP, carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) and all chemicals were from Sigma-Aldrich, USA. Lectins (WGA, PHA, LCA) and LY294002 were purchased from Merck Millipore, Germany. 2(trifluoromethyl)phenothiazine (2TFP), specific inhibitor of Nox1 [30 (link)], was a gift of Prof. Bruno Tasso Dept. PHArmacy, Genoa University, Genoa. Apocynin, DPI, DTT, LY294002, piceatannol, PP2, 2TFP and FCCP, were diluted in saline from a stock DMSO solution immediately before each experiment. MitoProbe™ Tetramethylrhodamine Methyl Ester (TMRM) from ThermoFisher Scientific was a gift from Dr. Paolo Degan, UO Mutagenesi e Prevenzione Oncologica, IRCCS Ospedale Policlinico San Martino, Genoa. ATP bioluminescence assay kit CLSII and ATP standard solution were from Roche, Switzerland.
6
Differentiation of Leishmania infantum Promastigotes to Amastigote-like Forms
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Late logarithmic phase cultures of L. infantum promastigotes were transferred to RPMI-1640 medium supplemented with 25% FBS and incubated at 37 °C (5% CO2) for 16–24 hr. Thereafter, the cells were centrifuged (1200×g at room temperature for 10 min) and resuspended in the same medium supplemented with (10 mM) succinate (Merck, Germany), titrated to pH 5.5 and incubated as above. Under this situation, promastigotes differentiated to amastigotes like form (AxAs) within 120 hr and amastigote forms without flagellate were produced (31 (link)-33 (link)).
7
Enhancing Erythroid Differentiation in MDS
8
Mitochondrial Function Assay Reagents
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Diethylenetriaminepentaacetic acid (DTPA, sodium salt), dimethyl sulfoxide (DMSO), ethylenediamine tetraacetic acid (EDTA), glucose, HCl, KCN, KH2PO4, Na2HPO4, NaH2PO4, NaCl, NaN3, sodium dodecyl sulphate, succinate, sucrose, and tris(hydroxymethyl)aminomethane (Tris) were obtained from Merck (Germany). Bovine serum albumin (BSA), decylubiquinone (dUQ), 2,6‐dichlorophenol–indophenol (DCPIP), dithiothreitol (DTT), cytochrome c3+ (cyt c3+), glutathione, hemin, hydroxyapatite, phenylmethylsulfonyl fluoride, penicillin–streptomycin solution, reduced nicotinamide–adenine dinucleotide (NADH), resazurin, sorbitol, Schneider's medium, brain heart infusion (BHI) medium, oligomycin (Oligo), antimycin A (AA), carbonyl cyanide‐m‐chlorophenylhydrazone (CCCP), paraffin oil, and Triton X‐100 were purchased from Sigma (USA). Triethanolamine hydrochloride was from Fluka (Switzerland), whereas Desferal (desferrioxamine mesylate [DFO]) was from Novartis Pharma (Germany), and 1‐hydroxy‐3‐methoxycarbonyl‐2,2,5,5‐tetramethylpyrrolidine‐HCl (CMH) was from Noxygen (Germany). Idebenone and zymolyase were from Takeda and Seikagaku Corporation (Japan), respectively. Yeast extract powder was supplied by Amresco (USA). 5‐Chloromethylfluorescein diacetate (CMFDA) was purchased from Abcam (USA).
9
Mitochondrial Respiration Assay in Cells
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A fixed number of cells were resuspended in MiR05 respiratory medium [20 mM HEPES (Thermo Fisher Scientific), 0.5 mM EGTA (Merck), 3 mM MgCl2⋅6H2O (Merck), 10 mM KH2PO4 (Merck), 20 mM taurine (Merck), 1 mg/ml bovine serum albumin (Merck), 60 mM potassium lactobionate (Merck), and 110 mM sucrose (Thermo Fisher Scientific), pH7.1] at a density of 1.5 x 105 cells⋅mL-1, and the suspension transferred to Oxygraph-2K (Oroboros Instruments, Innsbruck, Austria) chambers. Cells were first permeabilized by injection of 1 μL of 10 mg⋅mL-1 digitonin (Sigma) in DMSO (final concentration of 5 μg⋅mL-1). A substrate-inhibitor titration was then performed, comprising sequential injection of 2 mM malate (Merck), 0.2 mM octanoyl l -carnitine (Tocris Bioscience, Bristol, UK), 10 mM ADP (Merck), 10 μM cytochrome c (Merck), 25 mM pyruvate (Merck), 10 mM glutamate (Merck), 10 mM succinate (Merck), 0.5 μM rotenone (Merck), and 2.5 μM antimycin A (Merck). Datlab v. 6.0 (Oroboros Instruments) was used for data acquisition and analysis.
10
Mitochondrial Respiration Assay in Liver
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Mitochondrial respiration from liver homogenates was determined using high-resolution FluoRespirometry (Oxygraph-2k, Oroboros, Innsbruck, Austria) as described previously9 (link). In brief, after Complex I (CI) inhibition by rotenone (0.5 µmol/L, Merck, USA), samples were stimulated with Complex II (CII)-specific substrate (succinate, 10 mM, Merck, USA) for measuring LEAK respiration (LEAKS). CII-linked oxidative phosphorylation (OXPHOS CII) was determined at a saturation concentration of ADP (5 mmol/L, Merck, USA). ATP synthase was inhibited by oligomycin (2.5 μmol/L) to assess LEAK respiration in a non-phosphorylating state (LEAKOmy). At the end of the protocol, mitochondrial respiration was blocked by Complex III inhibitor Antimycin A (2.5 μmol/L, Merck, USA) to evaluate residual oxygen consumption (ROX). Mitochondrial oxygen consumption (volume-specific flux, JO2) was expressed in pmol/s/mL.
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