EnzyChrom™ L-Lactate Assay Kit

Application

• For quantitative determination of L-lactate (L-lactic acid) and evaluation of drug effects on its metabolism.

Key Features

• Sensitive and accurate. The 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 a 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

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%.

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.

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, 2×10⁶ cells to determine optimal cell number to be used in subsequent assays.

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.

Do samples containing high levels of pyruvate interfere with the assay?

Yes, high levels of pyruvate (>500µM) will interfere with the assay.

Chen, Z.-T., et al (2020). Long non-coding RNA Linc00092 inhibits cardiac fibroblast activation by altering glycolysis in an ERK-dependent manner. Cellular Signalling, 74, 109708. Assay: L-Lactate in cell media.

Domingo-Vidal, M., et al (2019). Cigarette smoke induces metabolic reprogramming of the tumor stroma in head and neck squamous cell carcinoma. Molecular Cancer Research: MCR, 17(9), 1893-1909. Assay: L-Lactate in fibroblast cell media.

Rolvien, T., et al (2020). Gnathodiaphyseal dysplasia is not recapitulated in a respective mouse model carrying a mutation of the Ano5 gene. Bone Reports, 12, 100281. Assay: L-Lactate in mouse serum.

Shao, S., et al (2020). Positive feedback in Cav-1-ROS signalling in PSCs mediates metabolic coupling between PSCs and tumour cells. Journal of Cellular and Molecular Medicine, 24(16), 9397-9408 Assay: L-Lactate in cell media.

Chu, J., et al (2020). Metabolic remodeling by TIGAR overexpression is a therapeutic target in esophageal squamous-cell carcinoma. Theranostics, 10(8), 3488-3502. Assay: L-Lactate in cell media.

Liu, H. T., et al (2019). HSF1: A mediator in metabolic alteration of hepatocellular carcinoma cells in cross-talking with tumor-associated macrophages. American Journal of Translational Research, 11(8), 5054-5064. Assay: L-Lactate in cell media.

Aranda-Diaz, A., et al (2020). Bacterial interspecies interactions modulate pH-mediated antibiotic tolerance. ELife, 9. Assay: L-Lactate in bacteria media.

Xue, H., et al (2020). Lactose-induced chronic diarrhea results from abnormal luminal microbial fermentation and disorder of ion transport in the colon. Frontiers in Physiology, 11, 877. Assay: L-Lactate in rat tissue.

Avnet, S., et al (2020). Benign albeit glycolytic: MCT4 expression and lactate release in giant cell tumour of bone. Bone, 134, 115302. Assay: L-Lactate in cell media, human tissue, and human serum.

Do, J. Y., et al (2020). Fursultiamine alleviates choroidal neovascularization by suppressing inflammation and metabolic reprogramming. Investigative Ophthalmology & Visual Science, 61(12), 24. Assay: L-Lactate in cell media.

Shin, H., et al (2020). Identification of aldh6a1 as a potential molecular signature in hepatocellular carcinoma via quantitative profiling of the mitochondrial proteome. Journal of Proteome Research, 19(4), 1684-1695. Assay: L-Lactate in human liver cell lysate.

Bawa, S., et al (2020). Drosophila TRIM32 cooperates with glycolytic enzymes to promote cell growth. ELife, 9. Assay: L-Lactate in fly larval tissue lysate.

Vargason, A. M., & Anselmo, A. C. (2020). Evaluation of surface modified live biotherapeutic products for oral delivery. ACS Biomaterials Science & Engineering. Assay: L-Lactate in bacteria media.

Chai, W., et al (2020). Decreased glycolysis induced dysfunction of NK cells in Henoch-Schonlein purpura patients. BMC Immunology, 21(1), 53. Assay: L-Lactate in cell media.

Martos-Rus, C., et al (2021). Macrophage and adipocyte interaction as a source of inflammation in kidney disease. Scientific Reports, 11(1), 2974. Assay: L-Lactate in cell media.

Bikash, C. R., & Tal-Gan, Y. (2020). Structure activity relationship study of the xip quorum sensing pheromone in streptococcus mutans reveal inhibitors of the competence regulon. ACS Chemical Biology, 15(10), 2833-2841. Assay: L-Lactate in bacteria media.

Li, X. G., et al. (2020). In vivo bioreactor using cellulose membrane benefit engineering cartilage by improving the chondrogenesis and modulating the immune response. Tissue Engineering and Regenerative Medicine, 17(2), 165-181. Assay: L-Lactate in cell bioreactor media.

Aranda-Diaz, Andres, et al (2019). Bacterial interspecies interactions modulate pH-mediated antibiotic tolerance in a model gut microbiota. bioRxiv: 538132. Assay: L-Lactate in bacteria culture.

Jung, Yi-deun, et al (2019). Lactate Activates the E2F Pathway to Promote Cell Motility by Up-Regulating Microtubule Modulating Genes. Cancers 11.3: 274. Assay: L-Lactate in human cells.

Liu, Na, et al (2019). Lactate inhibits ATP6V0d2 expression in tumor-associated macrophages to promote HIF-2alpha-mediated tumor progression. The Journal of clinical investigation 129.2. Assay: L-Lactate in mouse cells.

Li, Yong, et al (2018). Large Intergenic Non-coding RNA-RoR Inhibits Aerobic Glycolysis of Glioblastoma Cells via Akt Pathway. Journal of Cancer 9.5: 880. Assay: L-Lactate in human cells.

Zhang, Guiming, et al (2018). MCT1 regulates aggressive and metabolic phenotypes in bladder cancer. Journal of Cancer 9.14: 2492. Assay: L-Lactate in human cells.

Das, Champak, et al (2017). Multiplexed and fully automated detection of metabolic biomarkers using microdialysis probe. Sensors and Actuators B: Chemical 238: 633-640. Assay: L-Lactate in lactate product.

Di Pompo, Gemma, et al (2017). Intratumoral acidosis fosters cancer-induced bone pain through the activation of the mesenchymal tumor-associated stroma in bone metastasis from breast carcinoma. Oncotarget 8.33: 54478. Assay: L-Lactate in human cells.

Li, Xiaoli, et al (2017). Mitochondrial pyruvate carrier function determines cell stemness and metabolic reprogramming in cancer cells. Oncotarget 8.28: 46363. Assay: L-Lactate in mouse cells.

Weis, Sebastian, et al (2017). Metabolic adaptation establishes disease tolerance to sepsis. Cell 169.7: 1263-1275. Assay: L-Lactate in mice serum.

Zhang, Li, et al (2017). Clk1-regulated aerobic glycolysis is involved in glioma chemoresistance. Journal of neurochemistry 142.4: 574-588. Assay: L-Lactate in human & murine cells.

Hodgson, Douglas M., et al (2016). Segregation of the anodic microbial communities in a microbial fuel cell cascade. Frontiers in microbiology 7: 699. Assay: L-Lactate in lignocellulose cells.

Muller, Werner EG, et al (2016). Mineralization of bone-related SaOS-2 cells under physiological hypoxic conditions. The FEBS journal 283.1: 74-87. Assay: L-Lactate in human cells.

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.

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.

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.

Aronoff, D.M., 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.

Devalaraja-Narashimha, K.Padanilam, B.J. (2009). PARP-1 inhibits glycolysis in ischemic kidneys. J Am Soc Nephrol 20(1):95-103. Assay: L-Lactate in mouse kidney.

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 media.

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.

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 media.

Milovanova, T.N., 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 matrigel plugs.

Roy, S.G., 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.

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.

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.

Zheng, Y., et al. (2009). Anergic T cells are metabolically anergic. J Immunol 183(10):6095-101. Assay: L-Lactate in mouse cell culture media.

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If you or your labs do not have the equipment or scientists necessary to run this assay, BioAssay Systems can perform the service for you.

– Fast turnaround
– Quality data
– Low cost

Please email or call 1-510-782-9988 x 2 to discuss your projects.