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. D-Lactate is produced in only minor quantities in animals and measuring for D-lactate in animal samples is a means to determine the presence of bacterial infection.
Simple, direct and automation-ready procedures for measuring lactate concentration are very desirable. 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 lex/em=530/585 nm, is proportional to the lactate concentration in the sample.
Applications:Direct Assays: D-lactate in serum, plasma, urine, cell media samples and other biological samples.
Key Features:Sensitive and accurate. Detection limit of 1uM and linearity up to 50uM D-lactate in 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.
Storage:Store all reagents at -20°C. Shelf life: 6 months after receipt
Precautions: reagents are for research use only. Normal precautions for laboratory reagents should be exercised while using the reagents. Please refer to Material Safety Data Sheet for detailed information.
Sample Preparation And Considerations:The following substances interfere and should be avoided in sample preparation: EDTA (>0.5mM), ascorbic acid, SDS (>0.2%), sodium azide, NP-40 (>1%) and Tween-20 (>1%). Samples suspected of having endogenous L-LDH enzyme activity (e.g. serum, plasma, culture medium with FBS, etc.) should be deproteinated using a 10 kDa spin filter (e.g. Microcon YM-10). Deproteinated serum should be diluted 3X with dH2O. Samples containing higher than 50uM pyruvate require an internal standard.
Protocol:1. Standard Curve. Prepare 1000ul 40uM D-lactate Premix by mixing 2ul 20mM Standard and 998ul distilled water. For cell culture samples, prepare 1000ul 40uM D-lactate Premix by mixing 2ul 20mM Standard and 998ul culture medium without serum. Dilute standard as follows
No Premix+H2O or Medium D-Lactate (uM)1 100ul+0ul 402 60ul+40ul 243 30ul+70ul 124 0ul+100ul 0Transfer 50ul standards into wells of a black, flat bottom 96-well plate. Samples. Add 50ul sample to two separate wells. Set up two reactions for each sample: one with added Enzyme A (Sample) and a No Enzyme A control (Sample Blank). Samples requiring an internal standard, will need three separate reactions: 1) Sample plus Standard, 2) Sample alone and 3) Sample Blank. For the internal standard first prepare 400ul 250uM D-lactate standard by mixing 5ul 20mM Standard and 395ul dH2O. For the Sample plus Standard well, add 5ul 250uM D-lactate and 45ul sample. For the Sample and Sample Blank wells, add 5ul dH2O and 45ul sample
2. Reagent Preparation. Spin the Enzyme tubes briefly before pipetting. For each Sample and Standard well, prepare Working Reagent by mixing 40ul Assay Buffer, 1ul Enzyme A, 1ul Enzyme B, 10ul NAD and 5ul Probe. Fresh reconstitution is recommended. For the Sample Blanks, the Working Reagent includes 40ul Assay Buffer, 1ul Enzyme B, 10ul NAD and 5ul Probe (NO Enzyme A)
3. Reaction. Add 50ul Working Reagent per reaction well quickly. Tap plate to mix briefly and thoroughly. Incubate for 60 min at RT protected from light
4. Read fluorescence lex/em=530/585 nm.
Calculation:Plot the D-lactate Standard Curve and determine its slope. The D-lactate concentration of the sample is computed as follows:[D-Lactate] =FSAMPLE–FBLANK× n(uM) Slope (uM-1)where FSAMPLE and FBLANK are the fluorescence intensity values of the Sample and Sample Blank respectively. Slope is the slope of the standard curve and n is the dilution factor (e.g. n=3 for serum samples). If an internal standard was needed, the sample D-lactate concentration is computed as follows:FSAMPLE–FBLANK [D-Lactate] =× 27.8 (uM) FSTANDARD–FSAMPLEwhere FSAMPLE and FBLANK are the fluorescence intensity values of the Sample and Sample Blank respectively and FSTANDARD is the fluorescence intensity value of the Sample plus Standard
Note: if the sample DF value is higher than the DF for 40uM D-lactate standard or greater than the DF for the internal standard, dilute the sample in water and repeat the assay. Multiply the results by the dilution factor.
Materials Required, But Not Provided:Pipetting (multi-channel) devices. Black, flat bottom 96-well plates and fluorescent plate reader capable of reading at lex/em=530/585 nm.
