LDH Cell Proliferation and Cytotoxicity Assay Kit

100T, 500T

Cell proliferation and cytotoxicity assays

LDH-dependent NADH / INT colorimetric assay

Absorbance: 490 nm.

Kit heading: -20 °C, dark. Components: A, B1, B5 and Stop Solution at 4 °C; B2, B3 and B4 at -20 °C, dark.

12 months

Lactate dehydrogenase (LDH) is present inside cells and cannot pass through the plasma membrane under normal conditions. When cells are damaged, the altered membrane permeability allows LDH to be released from the cells into the culture medium. In the process of catalyzing lactate, the released LDH converts the oxidized form of nicotinamide adenine dinucleotide (NAD⁺) into the reduced form (NADH); NADH then reduces iodonitrotetrazolium chloride (INT) to a colored formazan compound via the hydrogen carrier phenazine methosulfate (PMS). This compound has an absorption peak at 490 nm, so cell damage (necrosis) can be assessed from the absorbance value. This product is for research use only and is not intended for clinical diagnosis.

(I) Preparation of the LDH substrate working solution

Before use, thaw all components at room temperature and mix them in the ratio B1 : B2 : B3 : B4 : B5 = 2 : 2 : 2 : 5 : 6 to obtain the LDH substrate working solution. Prepare fresh immediately before use.

(II) Procedure

1. Cell proliferation assay:

a. Collect cells in the logarithmic growth phase, adjust the concentration of the cell suspension according to the cell size and growth rate to set up a cell concentration gradient, and seed into a 96-well plate at 100 µL per well. Incubate at 37 °C and 5% CO₂ to allow the cells to adhere, for 6–24 hours.

b. Aspirate the culture medium and wash once with PBS. Replace with 200 µL of fresh culture medium (low-serum medium containing 1% serum, or suitable serum-free medium, is recommended), add 20 µL of Lysis Buffer per well, and pipette up and down several times to mix.

c. Continue incubating at 37 °C and 5% CO₂ for 30 min.

d. Centrifuge the culture plates at 1500 rpm for 5 min in a microplate centrifuge (if no microplate centrifuge is available, the supernatants may be transferred to centrifuge tubes and centrifuged). Transfer 120 µL of the supernatant from each well into the corresponding wells of a new 96-well plate, then add 100 µL of LDH substrate working solution to each well, mix, and let stand at room temperature protected from light for 10–30 min; alternatively, wrap the plate in aluminum foil and rock it slowly on a horizontal or orbital shaker for 10–30 min.

e. Add 30 µL of Stop Solution to each well and measure the absorbance at 490 nm (plot the absorbance on the Y-axis and the cell concentration on the X-axis).

2. Cytotoxicity assay:

(1) Experimental controls:

a. Background blank control: medium only (no cells), used to subtract the background readings caused by the LDH activity intrinsic to the culture medium and serum.

b. Sample control (spontaneous release): cells not treated with the drug, reflecting the level of LDH release caused by natural cell death.

c. Sample maximum enzyme activity control (maximum release): cells not treated with the drug but lysed with Lysis Buffer before measurement, representing the total amount of LDH detectable when 100% of the cells in the well are dead.

(2) Workflow:

a. Collect cells in the logarithmic growth phase, adjust the concentration of the cell suspension according to the cell size and growth rate, and seed into a 96-well plate at 200 µL per well. Incubate at 37 °C and 5% CO₂ to allow the cells to adhere for 6–24 hours, then treat with the drug at different concentrations.

b. At a defined time before measurement (e.g., 1 hour), aspirate the supernatant from the “sample maximum enzyme activity control” wells and replace with 200 µL of fresh culture medium (low-serum medium containing 1% serum, or suitable serum-free medium, is recommended); add 20 µL of Lysis Buffer per well and pipette up and down several times to mix.

c. Continue incubating at 37 °C and 5% CO₂ for 30 min.

d. Centrifuge each group of culture plates at 1500 rpm for 5 min in a microplate centrifuge (if no microplate centrifuge is available, the supernatants may be transferred to centrifuge tubes and centrifuged). Transfer 120 µL of the supernatant from each well into the corresponding wells of a new 96-well plate, then add 100 µL of LDH substrate working solution to each well, mix, and let stand at room temperature protected from light for 10–30 min; alternatively, wrap the plate in aluminum foil and rock it slowly on a horizontal or orbital shaker for 10–30 min.

e. Add 30 µL of Stop Solution to each well and measure the absorbance at 490 nm (a cytotoxicity curve can be plotted with the measured absorbance on the Y-axis and the drug concentration on the X-axis).

3. Calculation (the absorbance of the background blank control wells should be subtracted from the measured absorbance of every group):

Cytotoxicity or mortality (%) = (OD of treated sample − OD of sample control) / (OD of maximum enzyme activity control − OD of sample control) × 100%

1. The amount of LDH differs among cell types. It is recommended to perform a preliminary experiment with several samples before the formal experiment to optimize the assay conditions.

2. When a 96-well plate is used for detection and the cell culture period is long, the outer ring of wells may be abandoned (fill them with PBS, water, or culture medium instead); alternatively, place the 96-well plate near the water reservoir inside the incubator to alleviate evaporation.

3. If LDH in the culture medium is to be measured, note that serum contains lactate dehydrogenase; it is recommended that the serum concentration not exceed 1%, and heat-inactivated serum should preferably be used.

4. When seeding cells, make sure the cell suspension is thoroughly mixed to prevent the cells from settling, which would result in unequal cell numbers among wells; mix again after seeding every few wells.

5. For your safety and health, please wear a laboratory coat and disposable gloves.

For research use only.