Description
Lactic acid is an important intermediate product in the metabolic processes of organisms, closely related to carbohydrate metabolism, lipid metabolism, protein metabolism, and intracellular energy metabolism. Lactic acid concentration is a key indicator for evaluating glycogen metabolism and aerobic metabolism. Abnormally high concentrations of lactic acid are associated with pathological conditions such as cancer, diabetes, and lactic acidosis.The detection principle of this kit is as follows: Lactate dehydrogenase catalyzes the conversion of L-lactate to pyruvate, simultaneously reducing NAD+ to NADH and H+. Further, through the hydrogen transfer action of 1-mPMS, WST-8 reacts to form a yellow, soluble formazan. The absorbance at 450 nm is measured to calculate the L-lactate content in the sample.Detection Range: 0.03-2 mMSensitivity: 0.03 mMApplicable Samples: Animal and plant tissues, cells, bacteria, serum (plasma), or other liquids.L1501211Component48T96TStorageL1501211ALactate Assay Buffer70 mL70 mL×22-8℃L1501211BLactate Dehydrogenase0.7 mL1.4 mL-20℃L1501211CLactate Dehydrogenase Cofactor0.5 mL1 mL-20℃L1501211DWST-8350 µL700 µL-20℃. Store in the dark.L1501211EEnhancer70 µL140 µL-20℃. Store in the dark.L1501211FL(+)-Lactate Standard (100 mM)50 µL100 µL-20℃Please check the quantity of each component before the experiment.An additional 10% of each component is provided beyond the specified volume for standard curve preparation or preliminary experiments.User-Provided Instruments and ReagentsTypeNameNotesInstrumentMicroplate ReaderCapable of measuring absorbance at 450 nm.Consumables96-well MicroplateStandard transparent plate.ReagentsPBS (pH 7.4)For washing samples.OthersHomogenizer (for tissue samples), incubator, ice machine, low-temperature centrifuge, adjustable pipettes and tipsUsing a multichannel pipette for large-scale detection can improve efficiency.Experimental Procedure1. Reagent PreparationReagent NameReagent PreparationPrecautionsLactate Assay BufferReady-to-use; equilibrate to room temperature before use.4℃保存 Store at 4°C.Lactate DehydrogenaseReady-to-use;Keep on ice during the experiment; store aliquots at -20°C.Lactate Dehydrogenase CofactorReady-to-use;Keep on ice during the experiment; store aliquots at -20°C.WST-8Ready-to-use;Keep on ice during the experiment; store aliquots at -20°C.EnhancerReady-to-use;Keep on ice protected from light during the experiment; store aliquots at -20°C protected from light.L(+)-Lactate Standard (100 mM)Equilibrate to room temperature before use.100 mM, store aliquots at -20°C.2. Standard PreparationAdd 20 µL of the 100 mM standard to 980 µL of Lactate Assay Buffer to prepare a 2 mM standard stock solution. Aliquot and store at -20°C for up to 6 months. Dilute the 2 mM standard stock solution sequentially with Lactate Assay Buffer to prepare standard working solutions with final concentrations of 1 mM, 0.5 mM, 0.25 mM, 0.125 mM, 0.0625 mM, and 0.03125 mM. Use Lactate Assay Buffer as the blank.Standard Working SolutionStandard (µL)Lactate Assay Buffer (µL)Concentration (mM)1200 µL of 2 mM022200 µL of 2 mM20013200 µL of 1 mM2000.54200 µL of 0.5 mM2000.255200 µL of 0.25 mM2000.1256200 µL of 0.125 mM2000.06257200 µL of 0.0625 mM2000.03125Blank020003. Sample PreparationNote: Fresh samples are recommended. If not used immediately, samples can be stored at -80°C for up to 1 month. NADH or NADPH present in cell or tissue extracts can create background for lactate assay. To remove NADH or NADPH background, an equal amount of sample can be assayed without lactate dehydrogenase, and the background reading should be subtracted from the lactate reading. Endogenous lactate dehydrogenase (LDH) can degrade lactate. Samples containing LDH (e.g., cell culture medium, cell or tissue lysates) should be processed using a 10 kDa MW cutoff ultrafiltration tube (centrifuge at 12,000 g, 4°C for 10 min; follow the filter instructions) to remove all proteins. Use the filtrate for detection, then store at -80°C.3.1 Animal/Plant Tissues: Weigh approximately 0.1 g of tissue sample, add 1 mL of Lactate Assay Buffer, and homogenize on ice. Centrifuge at 12,000 g, 4°C for 5 min. Transfer the supernatant to a new tube and keep on ice for detection.3.2 Cells or Bacteria: Collect 5×10^6 cells. Wash the cells or bacteria with pre-cooled PBS. Centrifuge at 800 g for 2 min, discard the supernatant. Add 1 mL of Lactate Assay Buffer, and disrupt using an ultrasonic homogenizer on ice for 5 min (power 20% or 200 W, ultrasonic 3 s, interval 7 s, repeat 30 times). Centrifuge at 12,000 g, 4°C for 5 min. Collect the supernatant and keep on ice for detection.3.3 Plasma and Serum (Other Biological Fluids): Detect directly.4. Experimental Steps4.1 Microplate Reader Preparation: Preheat for at least 30 minutes, set wavelength to 450 nm.4.2 Working Reagent Preparation: 50 µL of Working Reagent is required per well. To avoid loss, prepare for 55 µL per single well system: Pipette 31 µL Lactate Assay Buffer, 8 µL Lactate Dehydrogenase Cofactor, 5 µL WST-8, 1 µL Enhancer, and 10 µL Lactate Dehydrogenase. Mix well. The Working Reagent must be prepared freshly and used immediately.4.3 Assay System Setup: Set up the detection system in the microplate according to the table below. The standard curve generally needs to be performed only once.ReagentStandard Well (µL)Test Well (µL)Sample050Standard Working Solution500Working Reagent50504.4 Absorbance Measurement: Mix well and incubate at 37°C protected from light for 30 min. Read the absorbance at 450 nm, recorded as Ablank, Astandard, and Atest. 5. Result CalculationThe following provides both the derived formula and the simplified calculation formula, which are completely equivalent.5.1 Data ProcessingCalculate ΔAstandard= Astandard- Ablank, ΔAtest = Atest - Ablank.5.2 Standard Curve PlottingPlot the standard curve with standard concentration as the y-axis and ΔAstandard as the x-axis. Substitute ΔAstandard into the equation to obtain the y value (mM).5.3 Sample L-Lactate Content Calculation① Calculated based on sample weight:L-Lactate (µmol/g) = y × Vsample ÷ (W × Vsample ÷ Vtotal) × n = y ÷ W × n② Calculated based on cell or bacterial count:L-Lactate (µmol/10⁴ cells) = y × Vsample ÷ (500 × Vsample ÷ Vtotal) × n = y ÷ 500 × n③ Calculated based on liquid volume:L-Lactate (mM) = y × Vsample ÷ Vsample × n = y × n④ Calculated based on protein concentration:L-Lactate (µmol/mg prot) = y × Vsample ÷ (Vsample × Cpr) × n = y ÷ Cpr × nParameter Description:1 mM = 1 mmol/L;Vsample : Volume of sample added, 0.05 mL;n: Sample dilution factor;Cpr: Sample protein concentration, mg/mL;W: Sample weight, g;Vtotal: Total volume of sample extract, 1 mL;500: Cell or bacterial count, 5×10⁶, converted to units of 10⁴.Result Presentation Using Previous Standard CurveTypical Standard Curve: y = 2.2613x - 0.0531Example-1: 50 µL of chicken serum was taken and processed according to the assay steps using a 96-well plate. The measured ΔAtest = Atest - Ablank= 0.435 - 0.096 = 0.339. Substituting into the standard curve, y = 0.713 mM. Calculated based on liquid volume: Lactate content (mM) = y × n = 0.713 × 5 = 3.565 mM.PrecautionsIt is recommended to perform preliminary experiments using 2-3 samples expected to have significant differences before formal testing.For tissue and cell samples, results can be normalized by measuring the protein concentration.This kit is compatible with spectrophotometer detection. Adjust the preparation volume of detection reagents proportionally according to the spectrophotometer's requirements.It is recommended to establish your own standard curve for improved accuracy. If not, you may refer to the typical standard curve formula provided in the results section for calculation.Biochemical reagents are generally irritating and biologically toxic. For your safety and health, please wear appropriate personal protective equipment (lab coat, mask, gloves, hair cap, etc.) throughout the experiment and perform experiments in a fume hood or biosafety cabinet.This product is for scientific research use only. Not intended for clinical diagnosis.Frequently Asked QuestionsWhat should I do if the sample ΔAtest is too high or too low?If the sample ΔAtest is >1.0, the lactate content in the sample is too high. Dilute the sample appropriately with Lactate Assay Buffer (multiply by the dilution factor in the calculation). If the sample ΔAtest is <0.13, increase the sample amount