Amplite™ Fluorimetric L-Lactate Assay Kit

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1e+41000100100101- Dose-responseData legend Generated with Quest Graph™ L-Lactate (uM) RFU Hover mouse to interact
L-lactate dose response was measured with Amplite™ Fluorimetric L-Lactate Assay Kit in a 96-well solid black plate using a Gemini (Molecular Devices) microplate reader.
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200 Tests 13814 $295


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Telephone: 1-800-990-8053
Fax: 1-408-733-1304
Email: sales@aatbio.com
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Overview

Ex/Em (nm)571/585
Storage Freeze (<-15 °C)
Minimize light exposure
InstrumentsFluorescence microplate reader
Category Cell Biology
Cell Metabolism
Related Redox Enzymes
Lactic acid is chiral and has two optical isomers: L-lactic acid and D-lactic acid. Lactate is constantly produced from pyruvate via the enzyme lactate dehydrogenase (LDH) in the process of metabolism and exercise. Monitoring lactate levels is a good way to evaluate the balance between tissue oxygen demand and utilization and is useful when studying cellular and animal physiology. D-lactate is not metabolized by mammals and its elimination from the body depends mainly on renal excretion. D- and L-lactic acid are found in many fermented milk products such as yoghurt and cheese, and also in pickled vegetables, and cured meats and fish. The D- and L-lactic acid (generated by bacteria) is a quality indicator of foods, such as egg, milk, fruit juice and wine. Abnormal high concentration of D-lactate in the blood is usually a reflection of bacterial overgrowth in the gastrointestinal tract. AAT Bioquest's Amplite™ Lactate Assay Kits (Cat# 13814 and 13815 for L-lactate assay, and Cat# 13810 and 13811 for D-lactate assay) provide both fluorescence and absorbance-based method for detecting either L-lactate or D-lactate in biological samples such as serum, plasma, urine, as well as in cell culture samples. In the enzyme coupled assay, lactate is proportionally related to NADH, which is specifically monitored by a fluorogenic NADH sensor. The signal can be read by a fluorescence microplate reader at Ex/Em = 540 nm/590 nm. With this Fluorimetric Amplite™ L-Lactate Assay Kit, we were able to detect as little as 1.4 µM L-lactate in a 100 µL reaction volume.




Protocol


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This protocol only provides a guideline, and should be modified according to your specific needs.
At a glance

Protocol summary

  1. Prepare L-lactate working solution (50 µL)
  2. Add L-lactate standards or test samples (50 µL)
  3. Incubate at room temperature for 30 min - 2 hours
  4. Monitor fluorescence increase at Ex/Em = 540/590 nm

Important notes
Thaw one vial of each kit component at room temperature before starting the experiment.

Key parameters
Instrument:Fluorescence microplate reader
Excitation:540 nm
Emission:590 nm
Cutoff:570 nm
Recommended plate:Solid black
Preparation of stock solutions
Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

1. NAD stock solution (100X):
Add 100 µL of H2O into the vial of NAD (Component C) to make 100X NAD stock solution.

2. L-Lactate standard solution (100 mM):
Add 200 µL of H2O or 1x PBS buffer into the vial of L-Lactate Standard (Component D) to make 100 mM L-Lacate standard solution.

Preparation of standard solution
L-Lactate standard

For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/13814

Add 10 µL of L-Lactate stock solution into 990 µL PBS buffer to generate 1 mM L-Lactate standard solution (Lac7). Take the 1 mM L-Lactate standard solution and perform 1:3 serial dilutions to get serial dilutions of L-Lactate standard (Lac6 - Lac1). Note: Diluted L-Lactate standard solution is unstable and should be used within 4 hours.

Preparation of working solution

Add 5 mL of Assay Buffer (Component B) into one bottle of Enzyme Probe (Component A). Add 50 µL NAD stock solution (100X) into the bottle of Component A, and mix well. Note: This L-lactate working solution is not stable, and should be used promptly.

Sample experimental protocol

Table 1. Layout of L-Lactate standards and test samples in a solid black 96-well microplate. Lac= L-Lactate Standards (Lac1 - Lac7, 1 µM to 1 mM), BL=Blank Control, TS=Test Samples. 

BL BL TS TS
Lac1 Lac1 ... ...
Lac2 Lac2 ... ...
Lac3 Lac3    
Lac4 Lac4    
Lac5 Lac5    
Lac6 Lac6    
Lac7 Lac7    

Table 2. Reagent composition for each well.

Well Volume Reagent
Lac1 - Lac7 50 µL Serial Dilutions (1 µM to 1 mM)
BL 50 µL Dilution Buffer
TS 50 µL Test Sample
  1. Prepare L-Lactate standards (Lac), blank controls (BL), and test samples (TS) according to the layout provided in Tables 1 and 2. For a 384-well plate, use 25 µL of reagent per well instead of 50 µL.

  2. Add 50 µL of L-Lactate working solution to each well of L-Lactate standard, blank control, and test samples to make the total L-Lactate assay volume of 100 µL/well. For a 384-well plate, add 25 µL of L-Lactate working solution into each well instead, for a total volume of 50 µL/well.

  3. Incubate the reaction at room temperature for 30 minutes to 2 hours, protected from light.

  4. Monitor the fluorescence increase with a fluorescence plate reader at Excitation = 530 - 570 nm, Emission = 590 - 600 nm (optimal Ex/Em = 540/590 nm, cut off at 570 nm).
Example data analysis and figures

The reading (RFU) obtained from the blank standard well is used as a negative control. Subtract this value from the other standards' readings to obtain the base-line corrected values. Then, plot the standards' readings to obtain a standard curve and equation. This equation can be used to calculate L-Lactate samples. We recommend using the Online Linear Regression Calculator which can be found at:

https://www.aatbio.com/tools/linear-logarithmic-semi-log-regression-online-calculator/

Figure 1. L-lactate dose response was measured with Amplite™ Fluorimetric L-Lactate Assay Kit in a 96-well solid black plate using a Gemini (Molecular Devices) microplate reader.

Disclaimer
AAT Bioquest provides high-quality reagents and materials for research use only. For proper handling of potentially hazardous chemicals, please consult the Safety Data Sheet (SDS) provided for the product. Chemical analysis and/or reverse engineering of any kit or its components is strictly prohibited without written permission from AAT Bioquest. Please call 408-733-1055 or email info@aatbio.com if you have any questions.





References & Citations

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The application of the improved 3D rat testicular cells co-culture model on the in vitro toxicity research of HZ1006
Authors: Xiaofang Zhang, Yuping Zhu, Yijun Tian, Han Yan, Lijun Ren, Wenjing Shi, Jiangbo Zhu, Tianbao Zhang
Journal: Drug and chemical toxicology (2018): 1--10

Arenobufagin inhibits prostate cancer epithelial-mesenchymal transition and metastasis by down-regulating β-catenin
Authors: Liping Chen, Weiqian Mai, Minfeng Chen, Jianyang Hu, Zhenjian Zhuo, Xueping Lei, Lijuan Deng, Junshan Liu, Nan Yao, Maohua Huang
Journal: Pharmacological Research (2017)

Fibroblast Activation Protein Alpha-Activated Tripeptide Bufadienolide Anti-tumor Prodrug with Reduced Cardiotoxicity
Authors: Li-Juan Deng, Long-Hai Wang, Cheng-Kang Peng, Yi-Bin Li, Mao-Hua Huang, Min-Feng Chen, Xue-Ping Lei, Ming Qi, Yun Cen, Wen-Cai Ye
Journal: Journal of Medicinal Chemistry (2017)

Neuronal Culture Microenvironments Determine Preferences in Bioenergetic Pathway Use
Authors: Juliane Sünwoldt, Bert Bosche, Andreas Meisel, Philipp Mergenthaler
Journal: Frontiers in Molecular Neuroscience (2017): 305

The use of KnockOut serum replacement (KSR) in three dimentional rat testicular cells co-culture model: An improved male reproductive toxicity testing system
Authors: Xiaofang Zhang, Lei Wang, Xiaodong Zhang, Lijun Ren, Wenjing Shi, Yijun Tian, Jiangbo Zhu, Tianbao Zhang
Journal: Food and Chemical Toxicology (2017)

High glucose and interleukin 1β-induced apoptosis in human umbilical vein endothelial cells involves in down-regulation of monocarboxylate transporter 4
Authors: Dong Wang, Qingjie Wang, Gaoliang Yan, Yong Qiao, Ling Sun, Boqian Zhu, Chengchun Tang, Yuchun Gu
Journal: Biochemical and biophysical research communications (2015): 607--614

Intracellular ATP decrease mediates NLRP3 inflammasome activation upon nigericin and crystal stimulation
Authors: Johji Nomura, Alexander So, Mizuho Tamura, Nathalie Busso
Journal: The Journal of Immunology (2015): 5718--5724






Additional Documents

 
Safety Data Sheet (SDS)


Catalogs
1. Enzyme Probes & Assay Kits

Application Notes
1. AssayWise Letters 2013, Vol 2(1)

Certificate of Analysis