Cell Titer Turbo 2D Luminescent Cell Viability Assay

1. Reagent preparation：
Reagent melt:The reagents were removed the day before the experiment and left at 4 °C overnight to melt. The reagents can also be taken out on the day of the experiment and melted at room temperature, or melted in a 22 °C water bath, butIt is necessary to note that the water temperature should not exceed25℃。
Equilibrate to room temperature:If the reagent melts at non-room temperature, it can be placed in a 22 ℃ water bath before use to ensure that the reagent is equilibrated to room temperature before being used for testing.
Note: Generally, it takes about 10 minutes for 5mL packaging; The 50 mL packaging takes about 20 minutes.
Mix well:Before useGentle upside down5TimeThe solution was mixed evenly.


2. Detection steps:
1. Cell culture: Use a 96-well plate suitable for chemiluminescence detection, and inoculate 100μL cells per well (determine the initial cell density according to the culture time, and the number of cells per well should not exceed 100,000 during detection). At the same time, set a well of culture medium containing no cells as a negative control. The concentration gradient of the cells can also be set to obtain the best experimental results. 37 °C, 5% CO₂Culture the cells and treat the cells with dosing at the appropriate time as needed.
2. (Optional) Preparation of ATP standard curve:
The prepared ATP standard solution was diluted with PBS to the appropriate concentration gradient, and 100 μL of the standard was added to each well of the 96-well plate.
3. Cell viability detection
(1) Thaw the frozen luminescence detection reagent, equilibrate it to room temperature (or 22 ℃ constant temperature water bath equilibrium), and take out the volume of detection reagent used for the current experiment
(2) Take out the cell culture plate and balance it at room temperature for 10 minutes (or balance it in a constant temperature water bath at 22 ℃, the time should not be too long, try to control it within 30 minutes)
(3) Add 100μL of detection reagent to each well of 96-well plate (due to the edge effect of the well, the luminescence signal may be unstable, so it is not recommended to plate at the edge)
(4) Shake at room temperature for 2 minutes to promote cell lysis;
(5) Place at room temperature for 10 minutes to stabilize the luminescent signal;
(6) Use a multifunctional microplate reader for chemiluminescence detection. Set the corresponding parameters according to the requirements of the instrument. The detection time of each hole is generally 0.25-1s. The specific adjustments need to be made according to the detection sensitivity of the instrument;
(7) Calculate the relative viability of cells according to the chemiluminescence reading, or calculate the ATP content according to the ATP standard curve to obtain the relative viability of cells.
Note: The detection effect varies with different types of cells. For some cells with particularly high ATP content, the chemiluminescence reading may continue to increase when the number of cells reaches more than 100,000, but the linear relationship is lost.

1, temperature:The reagent contains luciferase, and repeated freezing and thawing will affect its activity. It is recommended to store at-20 ℃ in the dark from light after dispensing. Reagents and cell samples should be balanced to room temperature before use to avoid the influence of enzyme catalytic effect;
2. Chemical factors:The reaction rate and luminescence intensity of luciferase are affected by the chemical environment. There are differences in luminescence intensity and attenuation rate among different types of media and serum. In addition, high drug content may interfere with the luciferase reaction, thus affecting the luminescence signal. It is recommended to set up cell culture medium control wells containing drug to eliminate solvent interference. If the culture medium has a great influence on the luminescence intensity, it can be removed before detection and washed once with PBS;
3. Light sensitivity:This reagent is sensitive to light and will accelerate the attenuation of luminous intensity if exposed to light during storage. If the reagent is transferred from the original container, make sure to keep it protected from light;
4. ATPContamination:It is recommended to wear a mask and latex gloves during operation and avoid contact with surfaces and equipment that may be contaminated. Avoid inserting the tip of the gun tip into the vial multiple times during operation.

I. Reagent preparation :
Reagent melting: The day before the experiment, the reagent was removed and placed at 4 ° C overnight to melt. The reagent can also be removed on the day of the experiment and melted at room temperature, or placed in a 22℃ water bath to melt, but need to note that the water temperature should not exceed 25℃.
equilibrate to room temperature: If the reagent melts under non-room temperature conditions, it can be placed in a 22℃ water bath before use to ensure that the reagent equilibrates to room temperature before being used for detection.
Note: Generally, it takes about 10 minutes for 5mL packaging; 50mL packaging takes about 20min.
Mix: gently invert 5 times to mix the solution well before use.
II. Detection steps:
1. Cell culture: use a 96-well plate suitable for chemiluminescence detection, and inoculate 100&mu in each well; L cells (according to the culture time to determine the initial seeding cell density, the number of cells per well should not exceed 100 000), at the same time, set up the culture medium without cells as a negative control. The concentration gradient of cells can also be set to obtain the best experimental results. Cells were cultured at 37 ° C with 5% CO₂and treated with drugs at the appropriate time as needed.
2. (Optional) Preparation of ATP standard curve:
The prepared ATP standard solution was diluted into an appropriate concentration gradient with PBS, and 100&mu was added to each well of a 96-well plate; L of the standard.
3, cell viability detection
(1) Thaw and thaw the luminescence method detection reagent, and balance to room temperature (or 22℃ constant temperature water bath equilibrium)
(2) Remove the cell culture plate, room temperature equilibrium for 10 minutes (or 22℃ constant temperature water bath equilibrium, the time should not be too long, Try to control within 30 minutes)
(3) Add 100&mu to each well of 96-well plate; L detection reagent (due to the edge effect of the well, it may cause the luminescence signal to be unstable, and it is not recommended to spread the plate at the edge)
(4) shake at room temperature for 2 minutes to promote the lysis of cells;
(5) place at room temperature for 10 minutes to make the luminescence signal tend to be stable;
(6) chemiluminescence detection was performed using a multifunctional microplate reader. Set the corresponding parameters according to the requirements of the instrument. The detection time of each well is generally 0.25-1s, and the specific need to be adjusted appropriately according to the detection sensitivity of the instrument.
(7) Relative cell viability was calculated according to the chemiluminescence reading, or ATP content was calculated according to the ATP standard curve to obtain the relative cell viability.
Note: The detection effect varies with the type of cells. For some cells with particularly high ATP content, the chemiluminescence reading may continue to increase when the number of cells reaches 100,000, but the linear relationship may be lost.

Store at-20 °C protected from light. In order to ensure the best performance, it is recommended to place it at-70 ℃ for long-term storage.