L-Lactate Assay Kit

K060

48T/96T

-20°C for 6 months, away from light.

Animal and Plant Tissues, Cells, Plasma, Serum or other Liquid samples.

Lactate is an important intermediate product in the metabolic process of organisms, which is closely related to glucose metabolism, lipid metabolism, protein metabolism and intracellular energy metabolism. The content of lactate is an important indicator to evaluate glycogen metabolism and aerobic metabolism. Abnormally high levels of lactate have been linked to pathological conditions such as cancer, diabetes and lactic acidosis. Lactate Assay Kit provides a convenient means for detecting L(+)-Lactate in biological samples such as animal and plant tissues, cells, serum, plasma or other liquid samples. In this kit, lactate is oxidized by lactate dehydrogenase to generate a product which interacts with a tetrazolium salt WST-8 dye to form a colorimetric (450 nm) product, proportional to the lactate present.

·Microplate reader or visible spectrophotometer capable of measuring absorbance at 450 nm

·Incubator, ice maker, freezing centrifuge

·96-well plate or microglass cuvette, precision pipettes, disposable pipette tips

·Homogenizer (for tissue samples)

Note: Briefly centrifuge small vials at low speed before opening.

Lactate Assay Buffer: Ready to use as supplied. Equilibrate to room temperature before use. Store at 4℃.

Lactate Dehydrogenase: Ready to use as supplied. Keep on ice and protect from light during the assay. Store aliquots at -20℃.

Lactate Dehydrogenase Cofactor: Ready to use as supplied. Keep on ice and protect from light during the assay. Store aliquots at -20℃.

WST-8: Ready to use as supplied. Keep on ice and protect from light during the assay. Store aliquots at -20℃, protected from light.

Reagent plus: Ready to use as supplied. Keep on ice and protect from light during the assay. Store aliquots at -20℃, protected from light.

Working Reagent: For each well, prepare 55 μL Working Reagent by mixing 31 μL Lactate Assay Buffer, 8 μL Lactate Dehydrogenase Cofactor, 5 μL WST-8, 1 μL Reagent plus and 10 μL Lactate Dehydrogenase, mix well. Working Reagent is freshly

prepared.

L(+)-Lactate Standard (2 mM): Dilute the Lactate Standard to 2 mM by adding 20 μL of the Lactate Standard to 980 μL of Lactate

Assay Buffer, mix well. Equilibrate to room temperature before use. Store aliquots at -20℃.

Setting of standard curves: Further dilute the 2 mM standard to 2, 1, 0.5, 0.25, 0.125, 0.0625, 0.0313 mM Standard with Lactate Assay Buffer, as shown in the following table.

Note: We recommend that you use fresh samples. If not assayed immediately, samples can be stored at -80℃ for one month.

1. Animal and Plant Tissues: The tissue was homogenized in Lactate Assay Buffer ice bath according to the proportion of 1 mL/0.1g. Centrifuge at 12,000 g for 5 min at 4℃. Use supernatant for assay, and place it on ice to be tested.

2. Cells: The cells were collected into the centrifuge tube, washed with cold PBS, the supernatant was discarded after centrifugation, and the cell 5 min was broken by Lactate Assay Buffer ice bath ultrasonic wave according to the proportion of 1 mL/5 million (power 20% or 200 W, ultrasonic 3 s, interval 7 s, repeat 30 times). Centrifuge at 12,000 g for 5 min at 4℃. Use

supernatant for assay, and place it on ice to be tested.

3. Plasma, Serum or other Liquid samples: Test directly.

Note: (1) NADH or NADPH from cell or tissue extracts generates background for the lactate assay. To remove the NADH or NADPH background, the same amount of sample can be tested in the absence of Lactate Dehydrogenase. Then the background readings can be subtracted from the lactate reading. (2) Endogenous Lactate Dehydrogenase (LDH) may degrade lactate. Samples containing LDH (such as culture medium or tissue lysate) should be filtered through a 10 kDaMW spin filter to remove all proteins and then kept at -80℃ for storage. (3) If the protein concentration of the sample is need to determined, it is recommended to use fn-test Cat #: K001 BCA Protein Assay Kit (protein quantification) to measure the protein concentration of the sample.

1. Preheat the microplate reader or visible spectrophotometer for more than 30 min, and adjust the wavelength to 450 nm. Visible spectrophotometer was returned to zero with deionized water.

2. Sample measurement. (The following operations are operated in the 96-well plate or microglass cuvette)

3. Mix well, Incubate for 30 min at 37℃ in the dark. The absorbance value is measured at 450 nm. The Blank Well is recorded as ABlank, the standard Well is marked as AStandard, and the test Well is marked as ATest. Finally calculate ΔATest=ATest-ABlank，ΔAStandard=AStandard-ABlank.

Note: In order to guarantee the accuracy of experimental results, need to do a pre-experiment with 2-3 samples. If ΔATest is less than 0.001, increase the sample quantity appropriately. If ΔATest is greater than 1.0, the sample can be appropriately diluted with Lactate Assay Buffer, the calculated result multiplied by the dilution factor, or decrease the sample quantity appropriately.

Note: We provide you with calculation formulae, including the derivation process and final formula. The two are exactly equal. It is suggested that the concise calculation formula in bold is final formula.

1. Drawing of standard curve

With the concentration of the standard solution as the y-axis and the ΔAStandard as the x-axis, draw the standard curve.

2. Calculation of Lactate content

Bring the ΔATest of the sample into the equation to get the y value (1 mM=1 μmol/mL)

(1) Calculated by fresh weight of samples

Lactate content (μmoL/g fresh weight)=y×V Sample÷(W×V Sample÷V Sample total)×n =y÷W×n

(2) Calculated by protein concentration

Lactate content (μmoL/mg prot)=y×V Sample÷(V Sample×Cpr)×n =y÷Cpr×n

(3) Calculated by volume of Liquid samples

Lactate content (μmoL/mL)=y×V Sample÷V Sample×n =y×n

(4) Calculated by number of cells

Lactate content (μmoL/10 4 cells)=y×V Sample÷(number of Cell×V Sample÷V Sample total)×n =y÷500×n

Where: VSample: add sample volume, 0.05 mL; W: weight of sample, g; VSample total: add Lactate Assay Buffer volume to sample, 1mL; n: the sample dilution factor; Cpr: sample protein concentration, mg/mL; 500: Total number of cells, 5×106.

Typical standard curve-data provided for demonstration purposes only. A new standard curve must be generated for each assay.