L-Lactic acid (L-LA) Content Assay Kit (WST-8, Micro Method) - BioReagent, high purity

Cat. No.: L1501211

GRADE & PURITY BioReagent

Overview

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 mM

Sensitivity: 0.03 mM

Applicable Samples: Animal and plant tissues, cells, bacteria, serum (plasma), or other liquids.

L1501211	Component	48T	96T	Storage
L1501211A	Lactate Assay Buffer	70 mL	70 mL×2	2-8℃
L1501211B	Lactate Dehydrogenase	0.7 mL	1.4 mL	-20℃
L1501211C	Lactate Dehydrogenase Cofactor	0.5 mL	1 mL	-20℃
L1501211D	WST-8	350 μL	700 μL	-20℃. Store in the dark.
L1501211E	Enhancer	70 μL	140 μL	-20℃. Store in the dark.
L1501211F	L(+)-Lactate Standard (100 mM)	50 μL	100 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 Reagents

Type	Name	Notes
Instrument	Microplate Reader	Capable of measuring absorbance at 450 nm.
Consumables	96-well Microplate	Standard transparent plate.
Reagents	PBS (pH 7.4)	For washing samples.
Others	Homogenizer (for tissue samples), incubator, ice machine, low-temperature centrifuge, adjustable pipettes and tips	Using a multichannel pipette for large-scale detection can improve efficiency.

Experimental Procedure

1. Reagent Preparation

Reagent Name	Reagent Preparation	Precautions
Lactate Assay Buffer	Ready-to-use; equilibrate to room temperature before use.	4℃保存 Store at 4°C.
Lactate Dehydrogenase	Ready-to-use;	Keep on ice during the experiment; store aliquots at -20°C.
Lactate Dehydrogenase Cofactor	Ready-to-use;	Keep on ice during the experiment; store aliquots at -20°C.
WST-8	Ready-to-use;	Keep on ice during the experiment; store aliquots at -20°C.
Enhancer	Ready-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 Preparation

Add 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 Solution	Standard (μL)	Lactate Assay Buffer (μL)	Concentration (mM)
1	200 μL of 2 mM	0	2
2	200 μL of 2 mM	200	1
3	200 μL of 1 mM	200	0.5
4	200 μL of 0.5 mM	200	0.25
5	200 μL of 0.25 mM	200	0.125
6	200 μL of 0.125 mM	200	0.0625
7	200 μL of 0.0625 mM	200	0.03125
Blank	0	200	0

3. Sample Preparation

Note: 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 Steps

4.1 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.

Reagent	Standard Well (μL)	Test Well (μL)
Sample	0	50
Standard Working Solution	50	0
Working Reagent	50	50

4.4 Absorbance Measurement: Mix well and incubate at 37°C protected from light for 30 min. Read the absorbance at 450 nm, recorded as A_blank, A_standard, and A_test .

5. Result Calculation

The following provides both the derived formula and the simplified calculation formula, which are completely equivalent.

5.1 Data Processing

Calculate ΔA_standard= A_standard- A_blank, ΔA_test = A_test - A_blank.

5.2 Standard Curve Plotting

Plot the standard curve with standard concentration as the y-axis and ΔA_standard as the x-axis. Substitute ΔA_standard into the equation to obtain the y value (mM).

5.3 Sample L-Lactate Content Calculation

① Calculated based on sample weight:

L-Lactate (μmol/g) = y × V_sample ÷ (W × V_sample ÷ V_total) × n = y ÷ W × n

② Calculated based on cell or bacterial count:

L-Lactate (μmol/10⁴ cells) = y × V_sample ÷ (500 × V_sample ÷ V_total) × n = y ÷ 500 × n

③ Calculated based on liquid volume:

L-Lactate (mM) = y × V_sample ÷ V_sample × n = y × n

④ Calculated based on protein concentration:

L-Lactate (μmol/mg prot) = y × V_sample ÷ (V_sample × Cpr) × n = y ÷ Cpr × n

Parameter Description:

1 mM = 1 mmol/L;

V_sample : Volume of sample added, 0.05 mL;

n: Sample dilution factor;

Cpr: Sample protein concentration, mg/mL;

W: Sample weight, g;

V_total: Total volume of sample extract, 1 mL;

500: Cell or bacterial count, 5×10⁶, converted to units of 10⁴.

Result Presentation Using Previous Standard Curve

Typical Standard Curve: y = 2.2613x - 0.0531

Example-1: 50 μL of chicken serum was taken and processed according to the assay steps using a 96-well plate. The measured ΔA_test = A_test - A_blank= 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.

Precautions

1. It is recommended to perform preliminary experiments using 2-3 samples expected to have significant differences before formal testing.

2. For tissue and cell samples, results can be normalized by measuring the protein concentration.

3. This kit is compatible with spectrophotometer detection. Adjust the preparation volume of detection reagents proportionally according to the spectrophotometer's requirements.

4. 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.

5. 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.

6. This product is for scientific research use only. Not intended for clinical diagnosis.

Frequently Asked Questions

What should I do if the sample ΔA_test is too high or too low?

If the sample ΔA_test 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 ΔA_test is <0.13, increase the sample amount.

Storage and Shipping

Storage

Store at 2-8°C,Protected from light,Store at -20°C

Shipped In

Ice chest + Ice pads

Stability And Storage

ach component has a shelf life of 6 months under corresponding storage conditions. L1501211A: Store at 2-8℃ long term (6 months). L1501211B, L1501211C, L1501211F: Store at -20℃ long term (6 months). L1501211D, L1501211E: Store at -20℃ long term (6 months