Self-supplied consumables:

Microplate reader or visible spectrophotometer (can measure the absorbance at 450 nm)

thermostat, ice machine, low temperature centrifuge

96-well plate or microglass cuvette, adjustable pipet gun and tip

deionized water

/>Homogenizer (if it is a tissue sample)

Reagent preparation

Note: Before opening the cap of each component (tubule reagent), please centrifuge at low speed.

1 × Assay Buffer: before use, Assay Buffer (5×) Dilute 5 times with deionized water to a concentration of 1× Assay Buffer, equilibrated to room temperature; Store at 4 ° C.

Lactate

ready-to-use; During the whole experiment, the ice was placed away from light; Aliquots were stored at -20 ° C in the dark.

NAD: ready-to-use; During the whole experiment, the ice was placed in the dark; Aliquots were stored at -20 ° C in the dark.

WST-8: ready-to-use type; During the whole experiment, the ice was placed away from light; Aliquots were stored at -20 ° C in the dark.

Enhancer

ready-to-use; Place on ice in the dark during the whole experiment; Aliquots were stored at -20 ° C in the dark.

Working Reagent

prepare 55 µ per well; L working solution, ready for use: absorb 31 μ L 1× Assay Buffer, 8 μ L NAD, 5 μ L WST-8, 1 μ L Enhancer & 10 μ L Lactate mix well.

Lactate Dehydrogenase Standard (20 U/mL): put 200μ L Lactate Dehydrogenase Standard with 800μ L 1× Assay Buffer was diluted to 20U/mL and mixed well; Place on ice in the dark; Store in aliquots at -20 ° C.

standard curve Settings: as indicated in the table below, with 1× Assay Buffer was used to dilute the 20 U/mL standard into 20, 16, 12, 8, 4, 2, and 1 U/mL standard solutions.

Sample preparation

Note

it is recommended to use fresh samples. If you don't use immediately, can be in the sample - 80 ℃ to save for a month.

1. Animal and plant tissues: weigh about 0.1 g of sample, add 1 mL of precooled 1×Assay Buffer, homogenize in an ice bath, centrifuge at 10,000 g at 4℃ for 15 min, take the supernatant and put it on ice for testing.

2, cells: Five million cells were collected into a centrifuge tube and washed with cold PBS. After centrifugation, the supernatant was discarded, and 1 mL of 1×Assay Buffer was added. The cells were sonicated in an ice bath for 5 min (20% power or 200 W, 3 s, 7 s interval, repeated 30 times). Then the samples were centrifuged at 10,000 g for 15 min at 4 ° C, and the supernatant was removed and placed on ice until measured.

3, serum, plasma and other liquid samples: direct determination.

Note

Protein quantification kit abs9232 (BCA assay) is recommended for protein concentration determination.

The experimental steps:

1. The microplate reader or visible spectrophotometer was preheated for more than 30 min, and the wavelength was adjusted to 450 nm. The visible spectrophotometer was zeroed with deionized water.
2. Operation table (the following operations are operated in 96-well plate or microglass cuvette) :

3, after mixing, 37 ℃ avoid light incubation for 30 min, measuring 450 nm absorbance, the blank hole for A short, standard hole as A standard, the determination of hole for A test. It was calculated that ΔA measured =A measured -A null, ΔA standard =A marked -A null.

Note

Before the experiment, it is recommended to select 2-3 samples with large expected differences for pre-experiment. If ΔA was less than 0.001, the sample size could be increased appropriately. If ΔA is greater than 2.0, the sample can be further diluted with 1×Assay Buffer, the calculated result multiplied by the dilution, or the sample size for extraction can be reduced.

The results were calculated as follows:

Note

we provide you with the formula, including the formula derivation and concise calculation formula. The two are exactly the same. The concise formula in bold is recommended as the final formula.

1, the drawing standard curve

The standard curve was plotted using the concentration of the standard solution as the Y-axis and ΔA as the X-axis.

2. Calculation of LDH content

The ΔA of the sample was substituted into the equation to obtain the y value (U/mL).

(1) Calculated according to fresh weight of sample

LDH content (U/g fresh weight)=y×V sample ÷(W×V sample ÷ total V sample)×n=y÷W×n

(2) calculated according to protein concentration

LDH content (U/mg protein)=y× V-like ÷(V-like ×Cpr)×n=y÷Cpr×n

(3) calculated by sample volume

LDH content (U/mL)=y×V sample ÷V sample ×n=y×n

(4) calculated by cell number

LDH content (U / 10 4 cells) = x y V sample present sample (500 x V present V sample total) (n = 500 x n y members present

V: join the sample volume, 0.05 mL; W: sample quality, g; V sample total: Add 1×Assay Buffer volume, 1 mL; n: sample dilution; Cpr: protein sample concentration, mg/mL; 500: total number of cells, 5 million.

Lactate dehydrogenase (LDH) is an oxidoreductase (EC1.1.1.27) that is found in a wide variety of organisms. It catalyzes the interconversion of pyruvate and lactate, which is accompanied by the interconversion of NADH and NAD+. Under hypoxic conditions, it converts pyruvate, the end product of glycolysis, to lactate. LDH quantification is clinically relevant because serum levels of certain LDH isoenzymes reflect pathological conditions in specific tissues. When disease, injury, or toxic substances damage tissues, the lactate dehydrogenase of the cells is released into the blood. Since lactate dehydrogenase is a fairly stable enzyme, it has been widely used to assess tissue and cell damage and toxicity. The CheKine? Lactate dehydrogenase (LDH) Activity Assay Kit (microassay) provides a simple and convenient microassay for the determination of lactate dehydrogenase in animal and plant tissues, cells, serum (plasma), and other biological fluids. In this trace method, LDH NAD reduction as NADH, NADH and WST - 8 interact to produce color, thus carries on the colorimetric determination of lambda (Max = 450 nm).