Cytotoxicity LDH Assay Kit

1. Control setup

High control: containing cells, culture medium, each sample adds 10 μL Lysis Solution, used to determine the maximum releasable quantity LDH of cells.
High blank control: containing culture medium, each sample adds 10 μL Lysis Solution, used to deduct the high control background absorbance value.
Low control: cells, culture medium, without lysis, used to measure spontaneous LDH release from untreated normal cells.
Background blank: containing culture medium only, used to deduct the background absorbance value of low control and sample wells.

Table 1. Control setup

2. General Protocol

2.1 Pre-experiment (optimization of cell number)

(1) After washing the cells with the medium, prepare the cell suspension of 5×10⁵ cells/ml.
(2) On a 96-well plate, add 100 μL of medium to each well.
(3) As shown in Figure 3, after adding 100 μL of cell suspension to row A of the 96-well plate (3 wells for high control and low control), Dilute the medium in 1/2 ratio with a multichannel pipette to form a serial cell gradient.
    In addition, prepare 3 wells each for a high control blank (medium + Lysis Solution) and a background blank (medium only).
(4) Incubate the plate at 37°C in a CO₂ incubator.
    * When incubate at 37°C in a CO₂ incubator for appropriate stage, ensure the incubation time setting is consistent with the cytotoxicity experiments.
(5) Add 10 μL of Lysis Solution to high control wells and high control blank wells, and add 10 μL of culture medium to low control wells and background blank wells.
(6) Incubate for another 30 min in a 37°C CO₂ incubator.
(7) Direct method: Remove 50 μL of the medium supernatant from each well, take the remaining medium and cells as the detection object, add 50 μL of Working Solution to each well, shake and mix well.
    Indirect method: Pipette 50 μL of supernatant from each well into a new 96-well plate. Then add 50 μL of Working Solution to each well of this 96-well plate, shake and mix.
    * To avoid aspirating the cells, aspirate the supernatant carefully.
    * The direct method is suitable for other experiments that do not need to collect live cells, and the indirect method is suitable for other experiments that need to collect live cells. Choose one of the methods to measure LDH activity according to the purpose of the experiment.
(8) Add 50 μL of Working Solution to each well, protect from light by wrapping aluminum foil, etc., and incubate at room temperature.
    * After adding Working Solution, the absorbance is proportional to the reaction time, it is recommended to detect within 0-30 min.
    * Due to the great difference between different cells, it is recommended to measure the absorbance at 0 min, 5 min, 10 min, 20 min and 30 min respectively before the first experiment to determine the best reaction time.
(9) After adding 50 μL of Stop Solution to each well, immediately measure the absorbance at 490 nm with a microplate reader.
    * Plot the absorbance as the X-axis and the cell concentration as the Y-axis, and select the best cell concentration according to the following requirements:
    - The difference between the OD. values of the high control and the low control >0.2;
    - The OD. value of this cell concentration on the linear curve is <2.0.

Fig 1. Example diagram of cell gradient configuration in pre-experiment on 96-well plate

2.2 Cytotoxicity assay

(1) Inoculate the cell suspension (100 μL/well) in a 96-well plate, and place the culture plate in an incubator for pre-culture for 24 hours.
(2) Add different concentrations of the treatment to be tested to the culture plate.
(3) Incubate the culture plate in the incubator for an appropriate time.
(4) Add 10 μL of Lysis Solution to the high control wells and high control blank wells, add 10 μL of medium to the low control wells, and incubate for 30 minutes in a 37°C CO₂ incubator.
(5) Direct method: Remove 50 μL of the medium supernatant from each well, take the remaining medium and cells as the detection object, add 50 μL of Working Solution to each well, shake and mix well.
    Indirect method: Pipette 50 μL of supernatant from each well into a new 96-well plate. Then add 50μL of Working Solution to each well of this new 96-well plate, shake and mix.
    * The direct method is suitable for other experiments that do not need to collect live cells, and the indirect method is suitable for other experiments that need to collect live cells. Choose one of the methods to measure LDH activity according to the purpose of the experiment.
(6) Incubate at room temperature in the dark.
    * The incubation time is the same as that of the pre-experiment.
(7) After adding 50 μL of Stop Solution to each well, immediately measure the absorbance at 490 nm with a microplate reader.
    * Use CCK-8 (GK10001) to obtain dead and live cell data.

2.3 Data processing

Cytotoxicity(%)=[(X-Z)/(Y-Z)] ×100%
      X: Absorbance value of sample well - absorbance value of background blank well
      Y: High control well absorbance value - high control blank well absorbance value
      Z: Absorbance value of low control well - absorbance value of background blank well

Fig 2. Analysis of cytotoxicity of mitomycin C on L-929 cells

3. Precautions

(1) Selection of 96-well plate: In the direct method, both adherent cells and suspension cells use a flat-bottom 96-well plate in the indirect method, a flat-bottom 96-well plate is used for adherent cells, and a round-bottom or V-bottom 96-well plate is used for suspension cells.
(2) When using a 96-well plate for detection, if the cell culture time is long, be sure to pay attention to the evaporation problem. On the one hand, since the circle around the 96-well plate is the easiest to evaporate, you can discard the circle and add the same amount of PBS, water or culture medium; on the other hand, you can place the 96-well plate close to the incubator Place within the water source to ease evaporation.
(3) Make sure that there are no air bubbles in each well before testing with a microplate reader, otherwise it will interfere with the measurement.
(4) A multi-channel pipette is recommended to reduce the variation between parallel wells.
(5) Since serum contains lactate dehydrogenase, it is recommended that the concentration of serum should not exceed 1%, and it is best to use heat-inactivated serum. If 10% serum must be used, be sure to set a background blank (media only) during detection to eliminate background.
(6) Factors such as excessive cell growth, high density, high centrifugation speed, and large temperature difference between inside and outside the incubator will cause an increase of lactate dehydrogenase release from cells.