EnzyFluo™ L-Lactate Assay Kit

Protocol SDS

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 1 µM and linearity up to 50 µM L-lactate in a 96-well plate assay.
• Convenient. The procedure involves adding a single working reagent and reading the fluorescence after 60 min. Room temperature assay.
• High-throughput. Can be readily automated as a high-throughput 96-well plate assay for thousands of samples per day.

Method

• FL530/585 nm

Samples

• Serum, plasma, cell culture media, etc

Species

• All

Procedure

• 60 min

Size

• 100 tests

Detection Limit

• 1 µM

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 EnzyFluo™ lactate assay kit is based on lactate dehydrogenase catalyzed oxidation of lactate, in which the formed NADH reduces a probe into a highly fluorescent product. The fluorescence intensity of this product, measured at λex/em = 530/585 nm, is proportional to the lactate concentration in the sample.

Does heparin, EDTA or citric acid interfere with assay?

EDTA and Heparin do not interfere. 10 mM 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?

Yes. To determine intracellular lactic acid concentrations, harvest cells (0.1-2 million cells per assay) in a 1.5mL 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. Prepare Working Reagent. Add 120 µL WR directly to the cell pellet and 80uL WR to standards in 96-wells. Vortex cell sample tube 1 min. Transfer 100 µL supernatant to 96-well. Read OD565nm kinetics on a plate reader.

Notes: 1. cell number. 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.
Working Reagent should contain a lysis reagent. BioAssay Systems will supply this reagent upon request.

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.

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 will interfere with the assay.

Sengupta, D., et al (2019). Multiplexed single-cell measurements of fdg uptake and lactate release using droplet microfluidics. Technology in Cancer Research & Treatment, 18, 1533033819841066. Assay: L-Lactate in human MDA-MB-231 breast cancer cells.

Vaccari Cardoso, B., et al (2021). Reducing l-lactate release from hippocampal astrocytes by intracellular oxidation increases novelty induced activity in mice. Glia. Assay: L-Lactate in HEK293 culture media.

Pires, S., et al (2020). Biological sex influences susceptibility to Acinetobacter baumannii pneumonia in mice. JCI Insight, 5(7). Assay: L-Lactate in mice bronchoalveolar lavage fluid.

Vaccari Cardoso, B. (2021). Novel molecular tools to selectively inhibit astrocyte-to-neurone L-lactate signalling. [Thesis] Assay: L-Lactate in HEK293 cells.

Rudnicki, Martina, et al (2018). Endothelial-specific FoxO1 depletion prevents obesity-related disorders by increasing vascular metabolism and growth. eLife 7: e39780. Assay: L-Lactate in mice serum.

Konrad, Csaba, et al (2017). Fibroblast bioenergetics to classify amyotrophic lateral sclerosis patients. Molecular neurodegeneration 12.1: 76. Assay: L-Lactate in human cells.

Palomino, Nahui, et al (2017). Vaginal Lactobacillus inhibits HIV-1 replication in human tissues ex vivo. Frontiers in microbiology 8: 906. Assay: L-Lactate in Lactobacillus culture.

Mongersun, Amy, et al. (2016). Droplet Microfluidic Platform for the Determination of Single-Cell Lactate Release. Analytical chemistry 88.6: 3257-3263. Assay: L-Lactate in unknown cancer cells.

Saville, Renee M., et al (2016). Compositions and methods for biological production of lactate from C1 compounds using lactate dehydrogenase transformants. U.S. Patent Application No. 14/898,948. Assay: L-Lactate in microorganism culture.

To find more recent publications, please click here.

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.