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

Cat. No.: D1501940

GRADE & PURITY BioReagent

Descripción general

  Lactic acid is a crucial intermediate in organism metabolism, closely related to carbohydrate, lipid, and protein metabolism, as well as cellular energy metabolism. Lactic acid content is an important 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. D-Lactic acid (D-LA) can be oxidized by D-lactate dehydrogenase, producing a product that interacts with the tetrazolium salt WST-8 dye to form a colored product proportional to the D-lactic acid concentration in the sample, with a maximum absorption peak at 450 nm.

  Detection Range: 0.031 - 1 μmol/mL
  Sensitivity: 0.031 μmol/mL
  Applicable Samples: Animal/plant tissues, cells, bacteria, serum (plasma)

Component	96T	Storage
Extraction Buffer	75 mL×2	2-8℃
ReagentⅠ	1.4 mL	-20℃
ReagentⅡ	1.2 mL	-20℃
ReagentⅢ	700 μL	-20℃. Store in the dark.
ReagentⅣ	140 μL	-20℃. Store in the dark.
Standard (100 μmol/mL)	100 μL	-20℃

Note: It is recommended to perform a pilot experiment with 2-3 samples expected to have significant differences before formal testing.

Required Materials and Equipment (Not Provided)

• Microplate reader or visible spectrophotometer (capable of measuring absorbance at 450 nm)

• 96-well plate or micro glass cuvette

• Adjustable pipettes and tips

• Incubator

• Ice maker

• Centrifuge

• Deionized water

• Homogenizer (for tissue samples)

Reagent Preparation

• Extraction Buffer: Ready-to-use. Equilibrate to room temperature (RT) before use. Store at 4°C.

• Reagent Ⅰ: Ready-to-use. Keep on ice throughout the experiment. Aliquot and store unused portions at -20°C. Avoid repeated freeze-thaw cycles.

• Reagent Ⅱ: Ready-to-use. Keep on ice throughout the experiment. Aliquot and store unused portions at -20°C. Avoid repeated freeze-thaw cycles.

• Reagent Ⅲ: Ready-to-use. Keep on ice protected from light throughout the experiment. Aliquot and store unused portions at -20°C protected from light. Avoid repeated freeze-thaw cycles.

• Reagent Ⅳ: Ready-to-use. Keep on ice protected from light throughout the experiment. Aliquot and store unused portions at -20°C protected from light. Avoid repeated freeze-thaw cycles.

• Working Reagent: Prepare fresh for immediate use. For each well, mix:

  • 31 μL Extraction Buffer

  • 8 μL Reagent Ⅱ

  • 5 μL Reagent Ⅲ

  • 1 μL Reagent Ⅳ

  • 10 μL Reagent Ⅰ

  • Mix thoroughly. (Total 55 μL per well required).

• Standard Solution Preparation:

  • Dilute the 100 μmol/mL Standard: Add 10 μL of Standard to 990 μL of Extraction Buffer to make a 1 μmol/mL stock solution. Mix well. Equilibrate to RT. Aliquot and store unused 100 μmol/mL Standard at -20°C.

  • Prepare standard curve points using the 1 μmol/mL stock as follows:

Standard Point	Volume of Dilution Source	Volume of Extraction Buffer (μL)	Final Concentration (μmol/mL)
Std.1	400µL 1 μmol/mL Standard	0	1
Std.2	200µL of Std.1 (1 μmol/mL)	200	0.5
Std.3	200µL of Std.2 (0.5 μmol/mL)	200	0.25
Std.4	200µL of Std.3 (0.25 μmol/mL)	200	0.125
Std.5	200µL of Std.4 (0.125 μmol/mL)	200	0.063
Std.6	200µL of Std.5 (0.063 μmol/mL)	200	0.031
Blank	0	200	0

Note: A standard curve must be generated with each experiment. Diluted standard solutions are unstable and must be used within 4 hours.

Sample Preparation

*Note: The use of fresh samples is recommended. If not used immediately, samples can be stored at -80°C for up to one month. Control the temperature and time during thawing. If thawed at room temperature, complete the process within 4 hours.*

1. Animal/Plant Tissues: Weigh approximately 0.1 g of tissue. Add 1 mL of pre-cooled Extraction Buffer and homogenize on ice. Centrifuge the homogenate at 12,000 g, 4°C for 5 min. Collect the supernatant and keep it on ice for assay.

2. Bacteria or Cells: Collect 5 million cells or bacteria by centrifugation. Wash the pellet with cold PBS, centrifuge, and discard the supernatant. Add 1 mL of Extraction Buffer. Disrupt the cells/bacteria by sonication on ice (5 min total, 20% power or 200W, pulse 3s on/7s off, repeat 30 times). Centrifuge the lysate at 12,000 g, 4°C for 5 min. Collect the supernatant and keep it on ice for assay.

3. Serum (Plasma): Assay directly.

Assay Procedure

1. Preheat the microplate reader or spectrophotometer for 30 min. Set the wavelength to 450 nm. Zero the spectrophotometer with deionized water.

2. Assay Setup (perform in a 96-well plate or micro glass cuvette):

Reagent	Test Well (μL)	Standard Well (μL)	Blank Well (μL)
Sample	50	0	0
Standard	0	50	0
Extraction Buffer	0	0	50
Working Reagent	50	50	50

3. Mix thoroughly and incubate at 37°C protected from light for 30 minutes.

Test, A_Standard, and A_Blank respectively. 5. Calculate ΔA_Test = A_Test - A_Blank and ΔA_Standard = A_Standard - A_Blank. *Note: The Blank and Standard wells need only be measured once per assay. It is advised to run a pilot test with 2-3 samples showing expected significant variation beforehand. If ΔA_Test is less than 0.03, consider increasing the sample amount. If ΔA_Test is greater than 1.0, dilute the sample further with Extraction Buffer (multiply the result by the dilution factor, n) or reduce the amount of sample used for extraction.* 

Result Calculation 

Note: Both the derived and simplified calculation formulas are provided and are equivalent. The simplified formulas (in bold) are recommended for final calculation. 

 Standard Curve: Plot the standard concentration (x-axis) against ΔA_Standard (y-axis) to obtain the standard curve equation. Substitute ΔA_Test into the equation to obtain x (μmol/mL). 

 D-Lactic Acid Content Calculation: 

 (1) Based on Sample Fresh Weight: 

D-LA content (μmol/g fresh weight) = x × V_Sample ÷ (W × V_Sample / V_Total) × n 

Simplified Formula: D-LA (μmol/g fresh weight) = x ÷ W × n

 (2) Based on Protein Concentration: 

D-LA content (μmol/mg prot) = x × V_Sample ÷ (V_Sample × Cpr) × n 

Simplified Formula: D-LA (μmol/mg prot) = x ÷ Cpr × n 

 (3) Based on Sample Volume:

D-LA content (μmol/mL) = x × V_Sample ÷ V_Sample × n 

Simplified Formula: D-LA (μmol/mL) = x × n 

 (4) Based on Cell/Bacteria Count: 

D-LA content (μmol/10⁴ cells) = x × V_Sample ÷ (500 × V_Sample / V_Total) × n 

Simplified Formula: D-LA (μmol/10⁴ cells) = x ÷ 500 × n 

 Parameter Description: 

 V_Sample: Sample volume added to the reaction = 0.05 mL 

 W: Sample mass (g) 

 V_Total: Total volume of Extraction Buffer added = 1 mL 

 n: Sample dilution factor 

 Cpr: Sample protein concentration (mg/mL) 

 500: Cell/Bacteria count (in ten-thousands: 500 × 10⁴ = 5 million) 

 Precautions 

This product is for scientific research use only. It is not intended for clinical diagnosis. For your safety and health, please wear a lab coat and disposable gloves during operation.

Almacenamiento y envío

Condiciones de almacenamiento de almacenamiento

Protected from light,Store at -20°C

Enviado en

Ice chest + Ice pads

Estabilidad y almacenamiento

Each component has a shelf life of 6 months under corresponding storage conditions.