Ldh activity was determined in tumor cell lysates by measuring the formation of soluble XTT formazan in direct relation to production of NADH over time at 475 nm at 37 °C using a SynergyMX plate reader (Biotek Instruments). Fresh tumor samples were homogenized with a tissue microgrinder followed by mechanical dissociation with a syringe and cell lysis in RIPA buffer (Pierce) with Halt protease and phosphatase inhibitors (Thermo-Fisher) on ice. After removing insoluble material by centrifugation at 8000g at 4 °C for 5 min, total protein concentration was determined using the BCA assay kit (Pierce) per manufacturer’s protocol with a microplate reader. Ten micrograms of protein was used per well for each tumor. Samples were run in triplicates. The staining solution contained 50 mM Tris buffer pH 7.4, 150 μM XTT (Sigma), 750 μM NAD (Sigma), 80 μM phenazine methosulfate (Sigma), and 10 mM of substrate lactate (Sigma). Ldh activity was determined in cell lysates by measuring the change in absorbance of their common substrate or product, NADH, over time at 340 nm at 25 °C using a Synergy-MX plate reader (Biotek Instruments).
Of substrate lactate
Manufactured by Merck Group
Lactate is a substrate commonly used in laboratory equipment and procedures. It serves as a source of energy and carbon for various cellular and biochemical processes. Lactate can be utilized by cells and enzymes for metabolic activities, but a detailed description of its specific functions or intended uses is not within the scope of this response.
Automatically generated - may contain errors
Lab products found in correlation
Phenazine methosulfate, by Merck Group (3 mentions)
Synergy mx plate reader, by Agilent Technologies (3 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Halt protease and phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
3 protocols using of substrate lactate
1
Quantifying Lactate Dehydrogenase Activity
2
Lactate Dehydrogenase (LDH) Activity Assay
3
Lactate Dehydrogenase (LDH) Activity Assay
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Ldh activity was determined in cell lysates by measuring the formation of soluble XTT formazan in direct relation to production of NADH over time at 475 nm at 37°C using a Synergy-MX plate reader (Biotek Instruments). Lysates were prepared in RIPA Buffer (Thermo Scientific Pierce). Protein content was determined using the BCA Protein Assay Kit (Thermo Scientific Pierce). 10 μg of protein were used per well. The staining solution contained 50 mM Tris buffer pH 7.4, 150 μM XTT (Sigma), 750 μM NAD (Sigma), 80 μM phenazine methosulfate (Sigma) and 10mM of substrate lactate (Sigma). Ldh activity was determined in cell lysates by measuring the change in absorbance of their common substrate or product, NADH, over time at 340 nm at 25°C using a Synergy-MX plate reader (Biotek Instruments).
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