For determination of necrosis, lactate dehydrogenase (LDH) was used as biomarker. Effluents of each group were collected for LDH assay over 3 min directly before global ischemia and four times over 3 min immediately after the start of reperfusion. LDH was assayed with the use of a commercially available assay (l -lactate dehydrogenase, Roche Applied Science, Mannheim, Germany) according to the manufacturer’s instructions. Optical density was measured in duplicate for each sample and multiplied by the respective coronary flow.
L lactate dehydrogenase
Manufactured by Roche
Sourced in Germany
L-lactate dehydrogenase is an enzyme that catalyzes the interconversion of lactate and pyruvate. It is involved in the process of glycolysis and has a key role in energy metabolism.
Automatically generated - may contain errors
Lab products found in correlation
Caspase glo 3 7 kit, by Promega (2 mentions)
Pyruvate kinase, by Merck Group (2 mentions)
L malic dehydrogenase, by Merck Group (2 mentions)
Glc 6 p, by Merck Group (2 mentions)
Adenosine diphosphate (adp), by Merck Group (2 mentions)
L malic acid, by Merck Group (2 mentions)
Adenosine triphosphate (atp), by Merck Group (2 mentions)
Hexokinase, by Roche (1 mentions)
Sodium ascorbate, by Merck Group (1 mentions)
Pyruvate, by Merck Group (1 mentions)
Rosiglitazone, by Cayman Chemical (1 mentions)
Carbonyl cyanide p trifluoromethoxyphenylhydrazone fccp, by Agilent Technologies (1 mentions)
Horseradish peroxidase conjugated goat anti rabbit, by Agilent Technologies (1 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions)
Sensifast sybr lo rox kit, by Meridian Bioscience (1 mentions)
Mission sirna, by Merck Group (1 mentions)
Hif 1α, by Cell Signaling Technology (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Collagenase type 2, by Merck Group (1 mentions)
9 protocols using l lactate dehydrogenase
1
Determination of Necrosis by LDH
2
Adipocyte Differentiation and Metabolism
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Collagenase type II (#C6885), Dulbecco’s Modified Eagle’s Medium (DMEM) (#D5030 and #D6429), dimethyl sulfoxide, glucose, 3-isobutyl-1-methylxanthine (IBMX), dexamethasone, isoproterenol (ISO), penicillin-streptomycin, pyruvate, MISSION siRNAs, sodium ascorbate and TRI Reagent were obtained from Sigma-Aldrich. Insulin was from Roche or kindly provided by Novo Nordisk A/S. glucose-6-phosphate dehydrogenase, hexokinase, L-lactate dehydrogenase, ATP and NADP+ and NAD+ were from Roche. DMEM (#52100), fetal bovine serum (FBS), Lipofectamine RNAiMAX, 10% newborn calf serum, L-glutamine and Opti-MEM I Reduced Serum Medium were obtained from Life Technologies. Rosiglitazone was from Cayman Chemical. SensiFAST SYBR Lo-ROX Kit was from Bioline. HEPES was from Lonza, and carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) was from Seahorse Bioscience. MitoQ was kindly provided by Dr. Mike Murphy44 (link). Primary antibodies were against HIF-1α (#14179, Cell Signaling Technology), UCP1 (#Ab10983, Abcam) and transcription factor II B (TFIIB) (#sc-225, Santa Cruz Biotechnology). The secondary antibody was horse radish peroxidase-conjugated goat anti-rabbit (#P0448, Dako).
3
Quantifying Cellular Metabolic Activity
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Glucose and lactate concentrations at each plating density were quantified in cell culture medium collected every 24 h. Glucose concentration was determined spectrophotometrically at 340 nm using the Glucose Assay Reagent (Sigma-Aldrich). Lactate concentration determination is based on the l -lactate-to-pyruvate transformation mediated by l -lactate dehydrogenase (Roche Diagnostics GmbH, Mannheim, Germany) in the presence of NAD+, with subsequent spectrophotometric quantification of the NADH co-product at 340 nm. Both glucose consumption and lactate production are expressed as the difference in the concentration in medium every 24 h normalized to cellular protein content, determined by SRB assay at the corresponding time point.
4
Native-PAGE Mobility Assay for ADP-Ribosylation
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Native-PAGE mobility assays were carried out in the same way as the fluorescence-based assays using non-fluorescent NAD+, 5 nM TccC3, and actin-binding reagents (final concentration are indicated in Figure 1 B and Figure S2 ) at 20 °C in Eppendorf tubes. The reactions were stopped by the addition of quenching buffer (10 mM Tris-HCl, pH 8.0, 100 mM L-lactate, 300 mM hydrazine, 60 U/mL L-lactate dehydrogenase (LDH from rabbit muscle; Roche, Mannheim, Germany)) [78 (link)]. Quenched reactions were resolved on 10% native polyacrylamide gels devoid of SDS and supplemented with 0.2 mM Ca2+ and 0.2 mM ATP. Gel electrophoresis was performed at 100 V using running buffer containing 25 mM Tris-HCl, 192 mM glycine, 0.1 mM Ca2+, 0.1 mM ATP, at 4 °C. Densitometry analysis of the bands corresponding to unmodified actin and ADPR-actin was performed using ImageJ software [79 (link)] to assess the degree of ADP-ribosylation and the activity of TccC3.
5
Synthesis and Purification of Protein
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All chemicals were used as supplied. Ethyl acetate, hexane, methanol, Petroleum ether 40–60 °C dichloromethane and magnesium sulphate were all purchased from Fisher Scientific at laboratory reagent grade. Deuterated chloroform (99.8 atom %D), dimethyl sulfoxide-d6 (99.9 atom %D), vinyl acetate (97.0%), 4,4′-azo-bis(4-cyanovaleric acid) (≥80.0%), 2,2′-azo-bis(2-methylpropionitrile) (98%), potassium ethyl xanthate (96%), 2-(methyl bromopropionate) (98%), aqueous hydrazine hydrate solution (50–60%), PBS buffer (preformulated tablets, yielding 0.01 M phosphate buffer, 0.0027 M potassium chloride and 0.137 M sodium chloride, pH 7.4), N-Ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC.HCl, >99.0%), N-hydroxy succinimide (NHS, 98%), imidazole (>99%), β-nicotinamide adenine dinucleotide, reduced disodium salt hydrate (NADH 97%), sodium pyruvate (ReagentPlus > 99%), polyethylene glycol (BioUltra 4000), poly(vinyl alcohol) (MW 9–10 kDa, 80% hydrolysed) and SealPlate films were purchased from Sigma Aldrich. L–lactate dehydrogenase was purchased from Roche. MilliQ water (18.2 mΩ).
Protein expression and purification are described in theSupporting Information .
Protein expression and purification are described in the
6
Measurement of LDH and Caspase 3/7 in RSV-Infected Cells
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LDH and caspase 3/7 were measured in the supernatants of RSV-infected HEp-2 and A549 cells as previously described (20 (link)). In brief, total LDH activity was measured in the supernatant using the LDH Cytotoxic Detection Kit Plus, (Roche Applied Science, Indianapolis, IN, USA) following protocol instructions. l- lactate dehydrogenase (Roche Applied Science) was used to construct a standard curve that demonstrated linear dynamic range (r = 0.998) at the dilutions tested from 3.9 to 125 milliunits per milliliter (mU/mL). Caspase 3/7, a marker of apoptosis, was measured using the Caspase-Glo-3/7 kit (Promega, Madison, WI, USA). The luminescence assay was measured using a Biotek Synergy H1 microplate reader (Biotek) and reported as relative luminescence units (RLU). Purified caspase 3 from ENZO (catalog no. BML-SE169) was used as the standard for Caspase Glo 3/7 assays and Gen5 Imager Software was used for analysis.
7
Measuring Cellular Injury and Apoptosis
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Total LDH activity was measured in apical wash and basolateral samples using a previously described method (22 (link)) . Apical wash and basolateral samples were assayed following protocol instructions (Cytotoxic Detection Kit, Roche Applied Science). To calculate absolute values, L-lactate dehydrogenase (Roche Applied Science) was used to construct a standard curve that demonstrated an ample linear dynamic range (r2≥0.998) at the dilutions tested from 0.8 to 110 milliunits per milliliter (mU/mL).
LDH release may be a marker of cell injury or death caused by virus or may be due to apoptosis induced by virus. To measure a marker of apoptosis in apical wash and basolateral samples, the Caspase-Glo-3/7 kit (Promega) was used as previously described (22 (link)). The luminescence of caspase was measured with a Synergy H1 microplate reader (Agilent Technologies) and expressed as relative luminescence units (RLU) and converted to units per milliliter (U/mL). A standard curve was constructed that demonstrated an ample linear dynamic range (r2>0.998).
LDH release may be a marker of cell injury or death caused by virus or may be due to apoptosis induced by virus. To measure a marker of apoptosis in apical wash and basolateral samples, the Caspase-Glo-3/7 kit (Promega) was used as previously described (22 (link)). The luminescence of caspase was measured with a Synergy H1 microplate reader (Agilent Technologies) and expressed as relative luminescence units (RLU) and converted to units per milliliter (U/mL). A standard curve was constructed that demonstrated an ample linear dynamic range (r2>0.998).
8
Enzymatic Assay for Metabolic Intermediates
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ATP, ADP, PEP, OAA, NADH, Glc6P, L-malic acid, pyruvate kinase, and L-malic dehydrogenase were from Sigma Aldrich. L-lactate dehydrogenase was from Roche. All other reagents were of the highest available quality.
9
Enzyme-Coupled Metabolite Assay
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ATP, ADP, PEP, OAA, NADH, Glc6P, L-malic acid, pyruvate kinase, and L-malic dehydrogenase were from Sigma Aldrich. L-Lactate dehydrogenase was from Roche. All other reagents were of the highest available quality.
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