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 Dlactate in animal samples is a means to determine the presence of bacterial infection. Simple, direct and automation-ready procedures for measuring lactate concentration are very desirable. BioAssay Systems' EnzyChromTM 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 565 nm, is proportionate to the lactate concentration in the sample.

Convenient. The procedure involves adding a single working reagent, and reading the optical density at time zero and at 20 min. Room temperature assay. No 37°C heater is needed.

High-throughput. Can be readily automated as a high-throughput 96- well plate assay for thousands of samples per day.

Precautions: 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.

Important: 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.

5. Calculation. Subtract OD0 from OD20 for the standard and sample wells. Use the DOD values to determine the sample D-lactate concentration from the standard curve. For samples requiring a No Enzyme A control, subtract the DODNoEnz value from the DODSample and use this DDOD value to determine the sample D-lactate concentration from the standard curve.

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. Simple, direct and automation-ready procedures for measuring lactate concentration are very desirable. BioAssay Systems' EnzyChromTM 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 565 nm, is proportionate to the lactate concentration in the sample.

Direct Assays: lactate in serum, plasma, and cell media samples.

Sensitive and accurate. Detection limit of 0.05 mM and linearity up to 2 mM L-Lactate in 96-well plate assay. For cell culture samples containing phenol red: detection limit of 0.1 mM and linearity up to 1 mM L-Lactate in 96-well plate assay. Convenient. The procedure involves adding a single working reagent, and reading the optical density at time zero and at 20 min. Room temperature assay. No 37°C heater is needed. High-throughput. Can be readily automated as a high-throughput 96- well plate assay for thousands of samples per day.

Assay Buffer: 10 mL NAD Solution: 1 mL

Enzyme A: 120 μL MTT Solution: 1.5 mL

Enzyme B: 120 μL Standard: 1.0 mL 20 mM L-Lactate

Storage conditions. Store all reagents at -20°C. Shelf life of at least 6 months (see expiry dates on labels).

Precautions: 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.

Important: 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.

1. Standard Curve. Prepare 1000 μL 2.0 mM L-lactate Premix by mixing 100 μL 20 mM Standard and 900 μL distilled water. For cell culture samples containing phenol red, prepare 1000 μL 1.0 mM lactate Premix by mixing 50 μL 20 mM Standard and 950 μL culture medium without serum. Dilute standard as follows. Transfer 20 μL standards into wells of a clear bottom 96-well plate.

1. Senadheera D et al (2009). Inactivation of VicK affects acid production and acid survival of Streptococcus mutans. J Bacteriol. 191(20):6415-24.

Colorimetric Kinetic Determination of Lactate Dehydrogenase Activity

LACTATE DEHYDROGENASE (LDH) is an oxidoreductase which catalyzes the interconversion of lactate and pyruvate. When disease or injury affects tissues containing LDH, the cells release LDH into the bloodstream, where it is identified in higher than normal levels. Therefore, LDH is most often measured to evaluate the presence of tissue or cell damage. The non-radioactive colorimetric LDH assay is based on the reduction of the tetrazolium salt MTT in a NADH-coupled enzymatic reaction to a reduced form of MTT which exhibits an absorption maximum at 565 nm. The intensity of the purple color formed is directly proportional to the enzyme activity.

Homogeneous and simple procedure. Simple “mix-and-measure” procedure allows reliable quantitation of LDH activity within 30 minutes.

Robust and amenable to HTS. All reagents are compatible with highthroughput liquid handling instruments.

Characterization and Quality Control for LDH production.

Drug Discovery: screen and evaluation of LDH modulators.

Precautions: 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.

This assay is based on a kinetic reaction. To ensure identical incubation time, addition of Working Reagent to samples should be quick and mixing should be brief but thorough. Use of a multi-channel pipettor is recommended. Assays can be executed at room temperature or 30°C.

Sample Preparation: Serum and plasma are assayed directly.

Tissue: prior to dissection, rinse tissue in phosphate buffered saline (pH 7.4) to remove blood. Homogenize tissue in 5 mL buffer containing 100 mM potassium phosphate (pH 7.0) and 2 mM EDTA, per gram tissue. Centrifuge at 10,000 x g for 15 min at 4°C. Remove supernatant for assay. Cell Lysate: collect cells by centrifugation at 2,000 x g for 5 min at 4°C. For adherent cells, do not harvest cells using proteolytic enzymes; rather use a rubber policeman. Homogenize or sonicate cells in an appropriate volume of cold buffer containing 100 mM potassium phosphate (pH 7.0) and 2 mM EDTA. Centrifuge at 10,000 x g for 15 min at 4°C. Remove supernatant for assay. All samples can be stored at –20 to –80°C for at least one month.

2. Pipet 950 μL Working Reagent to samples. Mix briefly.