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L lactate assay kit

Manufactured by Eton Bioscience
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The L-Lactate Assay Kit is a colorimetric assay designed to quantify the concentration of L-lactate in various biological samples, including cell culture media, tissue extracts, and body fluids. The kit utilizes an enzymatic reaction that produces a colored product, which can be measured spectrophotometrically. The assay is simple, sensitive, and can be performed in a 96-well format.

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23 protocols using l lactate assay kit

1

Quantifying Cellular Lactate Levels

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The cellular lactate level was measured using an l-lactate assay kit (Eton Bioscience, Charlestown, MA) according to the manufacturer’s recommendations. Briefly, an l-lactate assay solution was added to cells grown in 96-well plates. After 30 min incubation at 37 °C, the reaction was stopped by adding 0.5 M acetic acid, and the absorbance was measured at 490 nm using a microplate reader (CLARIOstar, BMG Labtech).
Welin Henriksson E., Wahren-Herlenius M., Lundberg I., Mellquist E, & Pettersson I. (1999). Key residues revealed in a major conformational epitope of the U1-70K protein. Proceedings of the National Academy of Sciences of the United States of America, 96(25), 14487-14492.
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2

Measuring L-Lactate Production in Cells

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Production of L-lactate was measured using the L-Lactate Assay Kit (Eton Bioscience, San Diego, CA). Briefly, the cells were treated with increasing concentrations of glucose, 2-DG and 3-BP for 24 hours. Next, 10 ul of the culture medium was transferred into a new 96-well plate; 40 ul of distilled water was subsequently added to each well. An additional 50 ul of glucose assay solution was added to each well, and the plates were incubated for 30 min at 37°C without CO2. Lactate levels were measured at a wavelength of 490 nm using a plate reader from Tecan. The experiments were performed in triplicate and repeated twice to assure consistency.
Sun L., Yin Y., Clark L.H., Sun W., Sullivan S.A., Tran A.Q., Han J., Zhang L., Guo H., Madugu E., Pan T., Jackson A.L., Kilgore J., Jones H.M., Gilliam T.P., Zhou C, & Bae-Jump V.L. (2017). Dual inhibition of glycolysis and glutaminolysis as a therapeutic strategy in the treatment of ovarian cancer. Oncotarget, 8(38), 63551-63561.
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3

Quantitative Analysis of Glucose and Lactate

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The culture medium was collected after SCC-9/DDP cells were incubated for 48 h in the six-well plate. Using a Glucose Assay Kit (Sigma-Aldrich) and a L-Lactate Assay Kit (Eton Bioscience, CA, USA), the concentration of glucose and lactate in spent medium was quantitatively measured. Glucose consumption of SCC-9/DDP cells was calculated as the glucose concentration of the unused cell culture medium minus the glucose concentration in the culture medium [32 (link)].
Liu X., Zhao T., Yuan Z, & Ge S. (2022). MIR600HG sponges miR-125a-5p to regulate glycometabolism and cisplatin resistance of oral squamous cell carcinoma cells via mediating RNF44. Cell Death Discovery, 8, 216.
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4

Measurement of Lactate Production

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Lactate production was measured by L-Lactate assay kit (Eton Bioscience Inc.) according to the manufaturer’s instructions. Simply, P493 cells were transfected with siP5CS or individual siPYCR1, 2 or L, and then cultured for 4 ds. 50 μl cell culture supernatant or L-lactate standard was added 50 μl L-lactate assay solution in 96-well flat bottom plate. The reaction mixtures were incubated for 30 mins at 37° incubator without CO2. 50 μl 0.5 M acetic acid was then added to stop the reaction. The absorbance at 490 nM was measured using a microplate reader. The measurements were normalized to living cell number of each group at the test day.
Liu W., Hancock C.N., Fischer J.W., Harman M, & Phang J.M. (2015). Proline biosynthesis augments tumor cell growth and aerobic glycolysis: involvement of pyridine nucleotides. Scientific Reports, 5, 17206.
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5

L-Lactate Assay in Cell Culture

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L-Lactate Assay Kit was purchased from Eton Bioscience Inc. and used per manufacturer’s instructions. Cells were plated at the same number and volume. Culture media were collected 72 h later and were diluted 1:10 with ddH2O prior to adding assay solution. Concentrations of lactate in samples were determined with the standard curve as a reference. Cell numbers counted at the time of medium harvest were used for normalization. For this assay, culture conditions of cells were not changed; the cells were cultured in RPMI-1640 media containing 20% fetal bovine serum (FBS) with 100 U/ml penicillin/streptomycin and 250 ng/ml amphotericin (PSA) (complete media).
Becker L.M., O’Connell J.T., Vo A.P., Cain M.P., Tampe D., Bizarro L., Sugimoto H., McGow A.K., Asara J.M., Lovisa S., McAndrews K.M., Zielinski R., Lorenzi P.L., Zeisberg M., Raza S., LeBleu V.S, & Kalluri R. (2020). Epigenetic Reprogramming of Cancer-Associated Fibroblasts Deregulates Glucose Metabolism and Facilitates Progression of Breast Cancer. Cell reports, 31(9), 107701.
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6

Metabolomic Analysis of CGNP Cultures

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1H NMR and LC-MS acquisition methods are described in detail in the Supplementary Materials and Methods. Briefly, CGNPs of each genotype were cultured in at least 3 replicate wells. Explanted CGNPs were maintained in [1,6-13C] glucose media for 24h followed by media sampling and cell extraction for metabolomic analysis. We normalized to cell number at 24 hours to account for differences in growth rates by counting cells in a replicate set of wells cultured in parallel.
For the enzymatic measurement of lactate, media from at least three replicate wells were sampled at the specified time points and lactate was quantified using the L-Lactate Assay Kit (#1200011002, Eton Bioscience, San Diego, CA, USA) per manufacturer’s protocol.
For PK activity assays, cells from at least three replicate wells per condition, or whole cerebella from at least three replicate mice per genotype, were lysed and processed for the colorimetric assay per manufacturer’s protocol (#K709-100, BioVision Inc., Milpitas, CA, USA).
Tech K., Tikunov A.P., Farooq H., Morrissy A.S., Meidinger J., Fish T., Green S.C., Liu H., Li Y., Mungall A.J., Moore R.A., Ma Y., Jones S.J., Marra M.A., Vander Heiden M.G., Taylor M.D., Macdonald J.M, & Gershon T.R. (2017). Pyruvate kinase inhibits proliferation during postnatal cerebellar neurogenesis and suppresses medulloblastoma formation. Cancer research, 77(12), 3217-3230.
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7

Glucose Uptake Measurement Protocol

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Cells were seeded in a 6 well plates at a density of 300,000 cells per well in DMEM complete medium. On the next day, the cells were washed twice in PBS and incubated in serum-glucose free medium for 2 hours. The medium was then removed, the cells were incubated for 1 hour in DMEM medium with 1g/L of glucose. The uptake was determined by using L-Lactate Assay Kit (Eton Bioscience) according to the manufacturer’s protocol.
Cosset É., Ilmjärv S., Dutoit V., Elliott K., von Schalscha T., Camargo M.F., Reiss A., Moroishi T., Seguin L., Gomez G., Moo J.S., Preynat-Seauve O., Krause K.H., Chneiweiss H., Sarkaria J.N., Guan K.L., Dietrich P.Y., Weis S.M., Mischel P.S, & Cheresh D.A. (2017). Glut3 addiction is a druggable vulnerability for a molecularly defined subpopulation of glioblastoma. Cancer cell, 32(6), 856-868.e5.
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8

Quantifying Glucose and Lactate Levels

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Liver cancer cells were plated in six-well plates and the culture medium were collected after incubation of cells for 48 h. The Glucose (GO) Assay kit (Sigma-Aldrich) and l-Lactate Assay kit (Eton Bioscience, San Diego, CA, USA) were then used to quantitatively measure the concentration of glucose and lactate in spent medium. Glucose consumption of liver cancer cells was obtained by using the formula: the glucose concentration of unused cell culture medium minus the glucose concentration in spent medium.
Jin X., Pan Y., Wang L., Zhang L., Ravichandran R., Potts P.R., Jiang J., Wu H, & Huang H. (2017). MAGE-TRIM28 complex promotes the Warburg effect and hepatocellular carcinoma progression by targeting FBP1 for degradation. Oncogenesis, 6(4), e312-.
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9

Lactate Measurement in Activated B Cells

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Purified B cells from healthy blood were cultured in medium or treated with CPG-DNA for 3 d. Thereafter, the culture supernatants were collected. In some cases, CD69+/hi and CD69− B cells purified from SLE were culture for 3 days. Concentrations of the lactate were determined using L-lactate assay Kit (eton bioscience). L-lactate assay Kit used are shown in Supplementary Table 5.
Wang X.Y., Wei Y., Hu B., Liao Y., Wang X., Wan W.H., Huang C.X., Mahabati M., Liu Z.Y., Qu J.R., Chen X.D., Chen D.P., Kuang D.M., Wang X.H, & Chen Y. (2022). c-Myc-driven glycolysis polarizes functional regulatory B cells that trigger pathogenic inflammatory responses. Signal Transduction and Targeted Therapy, 7, 105.
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10

Quantifying Lactate Secretion in Cell Lines

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6 × 105 cells were seeded in 6 cm plates, and 24 h later B16F10 (Mock or CAV1) and HT29(US) (Mock or CAV1) cells were treated with IPTG 1 mM for 48 or 24 h, respectively, or MDA-MB-231 (shCAV1 o shC). After this time, culture media were collected and diluted in PBS (1:2, 1:4, and 1:8), and cells were left untreated for 24 h. Absorbance was measured at 490 nm according to the instructions provided by the manufacturer (Eton Bioscience Inc.) of the L-Lactate assay kit.
Díaz-Valdivia N., Simón L., Díaz J., Martinez-Meza S., Contreras P., Burgos-Ravanal R., Pérez V.I., Frei B., Leyton L, & Quest A.F. (2022). Mitochondrial Dysfunction and the Glycolytic Switch Induced by Caveolin-1 Phosphorylation Promote Cancer Cell Migration, Invasion, and Metastasis. Cancers, 14(12), 2862.
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