Cytotoxic Drug Inhibitory Activity Assay: Hela cells were seeded into white 96-well plates with transparent bottoms at 5,000 cells per well, and treated with docetaxel at different concentrations. The plates were removed after 65 hours, and sequential fluorescent cell viability assay - CTG Cell Viability Assay was performed with the volume ratio of 60 μl medium - 60 μl fluorescent assay reagent - 60 μl CTG assay reagent. The reading of the well without drug treatment was set as 100%, and the percent reading of drug-treated wells was calculated to plot the curve and obtain the IC50 value.
A and B represent the fluorescent assay results of foreign similar brand products and UA CTF, respectively; C and D represent the CTG assay results after CTF assays of A and B, respectively. The IC50 values obtained by the two kits and two detection methods were very close, ranging from 6 to 9 nM. The results indicate that: the fluorescent cell viability assay reagent is compatible with the CTG assay reagent; the performance of CTF assay reagent is similar to that of foreign similar brand products.
Product Details
Product Details
Product Specification
| Synonyms | Fluorescent Cell Viability Assay Kit |
| Stability & Storage |
Store at -20℃ or below. The validity period is 12 months. |
Background
There are various methods for cell viability detection, such as detecting dye exclusion, changes in ATP concentration, and changes in enzyme activity. Cell detection sometimes requires research on the mechanism of cytotoxicity and/or the establishment of internal reference controls, which gives rise to the need for multiplex analysis. That is, the same sample is detected using multiple analytical methods with different detection mechanisms that do not interfere with each other. This allows for relevant comparisons to reveal the mechanism of cytotoxicity or exclude system differences. The detection of cell viability by our company's Fluorescent Cell Viability Assay Kit (UA-Glo® Fluorescent Cell Viability Assay) is based on the fact that proteases present only in living cells degrade peptide-fluorophore substrates to produce fluorescent signals. When the integrity of the cell membrane is impaired, these proteases lose their activity and no fluorescent signals are generated. The UA-Glo® Fluorescent Cell Viability Assay has a different detection principle from our company's UA-Glo® Luminescent Cell Viability Assay (Catalog No. UA070103) and can detect milder cell damage at an earlier stage. The two assays are compatible for multiplex detection. The UA-Glo® Fluorescent Cell Viability Assay is also compatible with our company's UA-Glo® Caspases 3/7 Cell Apoptosis Detection Kit (Catalog No. UA079012), enabling multiplex detection to determine the mechanism of cytotoxicity.
Components
Protease substrates and buffer solutions are mixed and filled into 10 ml or 100 ml brown bottles and 2 ml tubes. The specifications are as follows:
|
specifications |
CTF substrate |
CTF buffer |
Detectable number of wells in 96-well plate |
Detectable number of wells in 384-well plates |
|
10 ml |
100μl |
10ml |
100 wells |
500 wells |
|
5X10ml |
5x100μl |
5x10ml |
500 wells |
2,500 wells |
|
2X50 ml |
2x500μl |
2x50ml |
1,000 wells |
5,000 wells |
Protocol
1. Cell Preparation
1) Seed the cells to be tested at an appropriate density in a 96-well or 384-well cell culture plate. It is recommended to use a black-framed transparent cell culture plate.
2) Add the test compound at an appropriate concentration to the wells of the cell plate. The concentration of organic solvent in the culture medium should be kept below 1%. Continue culturing for an appropriate time according to the experimental requirements.
2. Cell Viability Assay
1) Take out the fluorescent cell viability assay reagent. After the CTF buffer is completely dissolved, equilibrate it to room temperature. Vortex the CTF substrate to mix it thoroughly, and centrifuge briefly to collect the CTF substrate at the bottom of the tube.
2) Prepare the CTF reaction solution: The CTF substrate is 100x. Prepare a 1x reaction solution with CTF buffer according to the required amount, and mix well.
3) Take out the cell culture plate to be tested, and add an equal volume of CTF reaction solution to each well. For example, add 100 μl of 1x CTF reaction solution to 100 μl of culture medium.
4) Shake the plate at low speed for 20 seconds to mix, and perform fluorescence detection after reacting at 37°C in the dark for 60 minutes. Different cells have different cleavage rates for the CTF substrate, so the optimal plate reading time needs to be optimized according to the cells. Plate reading can be done as early as 30 minutes after adding the reagent. Generally, the signal-to-noise ratio of the results read 60 minutes or longer is higher. It is recommended that the maximum plate reading time does not exceed 3 hours.
5) Read the fluorescence signal on a fluorescence microplate reader with excitation light (Ex) of 380-400 nm and emission light (Em) of 505 nm.
6) If needed, proceed with downstream multiplex analysis, such as Caspases 3/7 cell apoptosis detection.
Guidelines
1. It is recommended to aliquot the substrate and store it at -20°C after initial use, and prepare the reaction solution freshly each time.
2. It is not recommended to arbitrarily change the amount of reaction reagents without strict verification.
Picture
Picture
Bioactivity
HEK293 cells were counted and aliquoted into 96-well culture plates with the number of cells per well as shown in the table. Fluorescent cell viability assays were performed using UA CTF and foreign similar brand products respectively according to the product instructions. The readings in the figure represent the mean of two replicate readings at 60 minutes. The results indicate that the detection sensitivities of the two kits are similar.
