EnzyChrom™ L-Lactate Assay Kit
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Application
For quantitative determination of L-lactate (L-lactic acid) and evaluation of drug effects on its metabolism.

Key Features
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.

Method
OD565nm

Samples
Serum, plasma, cell culture media etc

Species
All

Procedure
20 min

Size
100 tests

Detection Limit
0.05 mM

Shelf Life
6 months

More Details
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’ EnzyChrom™ 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.

1. Does heparin, EDTA or citric acid interfere with assay?



EDTA and Heparin do not interfere. 10mM Citric acid lowers signal of 2 mM lactate by 15%.

2. Can this assay be used to determine lactate in cell culture supernatants that contain phenol red?



Yes, lactate assays can be performed in culture media that contains phenol red. We recommend diluting the standard in the control medium (i.e. that does not contain lactate). The detection range is 74 µM to 1 mM. If the lactate concentration in a sample is higher than 1 mM, dilute sample in the medium and repeat assays. Multiply the results by the dilution factor. The detection limit is around 74 µM. Follow the following table for standard dilutions.

3. Can this kit be used to measure lactic acid concentrations in cells?



To determine intracellular lactic acid concentrations, harvest cells (0.1-2 million cells per assay) in a 1.5-mL tube, centrifuge 1-2 min at 3,000 rpm on a table centrifuge. Remove culture medium, wash cells quickly with cold PBS. Immediately remove any PBS. Lyse cells in dH2O by homogenization, ultrasonic treatment or freeze-thaw. Centrifuge cells to obtain supernatant and follow the protocol for lactic acid determination.

Alternatively, cells may be lysed with the Working Reagent containing 0.1% Triton X-100 (WR/Triton). Add 120 µL WR/Triton directly to the cell pellet tubes and 80 μL WR/Triton to standards in 96-wells. Vortex cell sample tubes 1 min to lyse cells. Transfer 100 µL supernatant to separate 96-wells. Read OD565nm kinetics on a plate reader.

Note: It is prudent to run several doses of cells, e.g. 0.1, 1, 2x106 cells to determine optimal cell number to be used in subsequent assays.

4. How to prepare tissue homogenates for use in the assay?



For tissue sample preparation, we recommend the following:

1. Homogenize tissue in phosphate buffered saline. This can be done using a homogenizer (e.g. the Dounce type) or a grinder. If you use a lysis buffer, the buffer should not contain ascorbic acid, SDS, sodium azide, NP-40 and Tween-20.

2. Centrifuge to pellet any debris. This can be done at room temperature in a table centrifuge (e.g. 5 min at 14,000 rpm). The supernatant should be clear.

3. Use the supernatant for lactate assays.

For more detailed product information and questions, please feel free to Contact Us. Or for more general information regarding our assays, please refer to our General Questions
1. Aronoff, DM et al (2009). E-prostanoid 3 receptor deletion improves pulmonary host defense and protects mice from death in severe Streptococcus pneumoniae infection. J Immunol 183(4):2642-9. Assay: L-Lactate in Mouse Plasma (Pubmed).

2. Zheng, Y et al (2009). Anergic T cells are metabolically anergic. J Immunol 183(10):6095-101. Assay: L-Lactate in Mouse Cell culture medium (T cells) (Pubmed).

3. Le Nihouannen, D et al (2009). Ascorbic acid accelerates osteoclast formation and death. Bone 46(5):1336-43. Assay: L-Lactate in Mouse Cell culture medium (RAW 264.7 monocytic cell line) (Pubmed).

4. Fan, Y et al (2009). Akt and c-Myc differentially activate cellular metabolic programs and prime cells to bioenergetic inhibition. J Biol Chem 285(10):7324-33. Assay: L-Lactate in Mouse Cell culture medium (FL5.12 pre B-cell line) (Pubmed).

5. Wei, S et al (2010). Energy restriction as an antitumor target of thiazolidinediones. J Biol Chem 285(13):9780-91. Assay: L-Lactate in Human LNCaP cells (Pubmed).

6. Toschi, A et al (2010). Phospholipase D-mTOR requirement for the Warburg effect in human cancer cells. Cancer Lett 299(1):72-9. Assay: L-Lactate in Human (renal and breast cancer cells) (Pubmed).

7. Milovanova, TN et al (2009). Hyperbaric oxygen stimulates vasculogenic stem cell growth and differentiation in vivo. J Appl Physiol 106(2):711-28. Assay: L-Lactate in N/A Matrigel plugs (Pubmed).

8. Devalaraja-Narashimha K, Padanilam BJ (2009). PARP-1 inhibits glycolysis in ischemic kidneys. J Am Soc Nephrol 20(1):95-103. Assay: L-Lactate in Mouse Kidney (Pubmed).

9. Kashimshetty, R et al (2009). Underlying mitochondrial dysfunction triggers flutamide-induced oxidative liver injury in a mouse model of idiosyncratic drug toxicity. Toxicol Appl Pharmacol 238(2):150-9. Assay: L-Lactate in Mouse Serum (Pubmed).

10. Weber, J et al (2009). Hyperalgesia induced by oral stavudine administration to rats does not depend on spinal neuronal cell death, or on spinal or systemic inflammatory cytokine secretion, or metabolic dysregulation. Neurotoxicology 30(3):423-9. Assay: L-Lactate in Rat Plasma (Pubmed).

11. Roy, SG et al (2009). Excess of glucocorticoid induces cardiac dysfunction via activating angiotensin II pathway. Cell Physiol Biochem 24(1-2):1-10. Assay: L-Lactate in Rat Serum (Pubmed).

12. Senadheera, D et al (2009). Inactivation of VicK affects acid production and acid survival of Streptococcus mutans. J Bacteriol 191(20):6415-24. Assay: L-Lactate in Streptococcus mutans Bacteria (Pubmed).

13. Krastel, K et al (2010). Characterization of a glutamate transporter operon, glnQHMP, in Streptococcus mutans and its role in acid tolerance. J Bacteriol 192(4):984-93. Assay: L-Lactate in Streptococcus mutans Bacteria culture supernatant (Pubmed).

To find more recent publications, please click here.
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EnzyChrom™ L-Lactate Assay Kit
Catalog No: ECLC-100
Price: $329    Qty:
For orders of 10 or more kits, please contact us at 1-510-782-9988 x 1 for best pricing.

Shipping: On Ice
Shipment: Same Day for order by 2pm PST
Delivery: 1-2 days (US), 3-6 days (Intl);
Storage: -20°C



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