D-Lactate Assay Kit, BioAssay(TM), Colorimetric

Lactate is generated by lactate dehydrogenase (LDH) under hypoxic or anaerobic conditions. Monitoring lactate levels is, therefore, a good indicator of the balance between tissue oxygen demand and utilization and is useful when studying cellular and animal physiology. D-lactate is produced in only minor quantities in animals and measuring for D-lactate in animal samples is a means to determine the presence of bacterial infection.\n\nIntended Use:\nSimple, direct and automation-ready procedures for measuring lactate concentration are very desirable. Lactate assay kit is based on lactate dehydrogenase catalyzed oxidation of lactate, in which the formed NADH reduces a formazan (MTT) Reagent. The intensity of the product color, measured at 565nm, is proportionate to the lactate concentration in the sample.\n\nMatrix:\nDirect Assays: D-lactate in serum, plasma, and cell media samples.\n\nDetection Range:\nDetection limit of 0.05mM and linearity up to 2mM D-lactate in 96-well plate assay. For cell culture samples containing phenol red: detection limit of 0.1mM and linearity up to 1mM D-lactate in 96-well plate assay.\n\nSimple Protocol:\nConvenient. The procedure involves adding a single working reagent, and reading the optical density at time zero and at 20 min. Room temperature assay. High-throughput. Can be readily automated as a high-throughput 96-well plate assay for thousands of samples per day.\n\nKit Contents: (100 tests in 96-well plates)\nAssay Buffer: 10ml NAD Solution: 1ml\nEnzyme A: 120ul MTT Solution: 1.5ml\nEnzyme B: 120ul Standard: 1.0ml 20mM D-lactate\nStorage conditions. Store all reagents at -20 degrees C. Shelf life: 6 months after receipt.\nPrecautions: reagents are for research use only. Normal precautions for laboratory reagents should be exercised while using the reagents. Please refer to Material Safety Data Sheet for detailed information.\n\nProtocol:\nImportant: this assay is based on an enzyme-catalyzed kinetic reaction. Addition of Working Reagent should be quick and mixing should be brief but thorough. Use of a multi-channel pipettor is recommended.\n1. Standard Curve. Prepare 1000ul 2.0mM D-lactate Premix by mixing 100ul 20mM Standard and 900ul distilled water. For cell culture samples containing phenol red, prepare 1000ul 1.0mM lactate Premix by mixing 50ul 20mM Standard and 950ul culture medium without serum. Dilute standard as follows. Transfer 20ul standards into wells of a clear bottom 96-well plate.\nNo Premix+H2O or Medium Vol (ul) D-lactate (mM)\n1 100ul+0ul 100 2.0 or 1.0\n2 80ul+20ul 100 1.6 or 0.8\n3 60ul+40ul 100 1.2 or 0.6\n4 40ul+60ul 100 0.8 or 0.4\n5 30ul+70ul 100 0.6 or 0.3\n6 20ul+80ul 100 0.4 or 0.2\n7 10ul+90ul 100 0.2 or 0.1\n8 0ul+100ul 100 0\nSamples. Add 20ul sample per well in separate wells. For samples with potential endogenous enzyme activity (i.e. serum, plasma, tissue extracts), two reactions should be run: one with added Enzyme A and a No Enzyme A control. Serum and Plasma should be diluted at least 2

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SPECIFICATIONS

Catalog Number

L1011-04

Size

1Kit

References

1. Babson, AL and Babson, SR. (1973) Kinetic Colorimetric Measurement of Serum Lactate Dehydrogenase Activity. Clin Chem. 19(7): 766-9.\n2. Karlsen RL, Norgaard L, Guldbrandsen EB (1981). A rapid method for the determination of urea stable lactate dehydrogenase on the 'Cobas Bio' centrifugal analyser.Scand J Clin Lab Invest. 41(5): 513-6.\n3. Coley HM, Lewandowicz G, Sargent JM, Verrill MW (1997). Chemosensitivity testing of fresh and continuous tumor cell cultures using lactate dehydrogenase. Anticancer Res. 17(1A): 231-6.