Cell viability is an indicator of the proportion of healthy living cells in a population, reflecting the overall health of cells. The determination of cell viability aims to evaluate the physical and metabolic state of cells to determine the effects of treatment or culture conditions on cell homeostasis; CCK-8, MTT and other methods are usually used for detection.
1. Experimental principle
CCK-8 is a highly sensitive, non-radioactive colorimetric detection method based on WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonic acid benzene)-2H-tetrazolyl monosodium salt] and is widely used to determine cell proliferation or cytotoxicity. The principle is that WST-8 is reduced to a highly water-soluble yellow formazan product (Formazan dye) by dehydrogenase in the cell mitochondria under the action of electron carriers. The amount of formazan product generated is proportional to the number of living cells; the more and faster the cell proliferation, the darker the color; the greater the cytotoxicity, the lighter the color. For the same cells, the depth of color is linearly related to the number of cells. Therefore, this characteristic can be used to directly analyze cell proliferation and toxicity.
2. Experimental principle diagram
2. Experimental steps
2.1 Cell plating treatment
2.1.1 Trypsinize the stable cells, count the cells, and adjust the cell concentration to 2×104/mL;
2.1.2 Inoculate 2×103 cells/well on a 96-well plate and culture the plate in an incubator for 24 h;
2.1.3 After treating the cells as needed, perform CCK8 detection at different times.
2.2 CCK8 detection of cell viability
2.2.1 Add CCK-8 solution to each well of the 24-well plate at 1 d, 4 d, and 7 d after cell inoculation;
2.2.2 Place the culture plate in a 37 ℃ incubator and incubate for 1 h;
2.2.3 After completion, transfer the reaction solution to a 96-well plate;
2.2.4 Set the microplate reader to 450 nm and detect the absorbance, and record the data.
3. Experimental results example
OD450 detection results under different serum concentration culture conditions
Group | Cell suspension volume/μL | Serum concentration/% | OD450 |
A | 200 | 10 | 1.91 ± 0.39 |
B | 100 | 10 | 1.75 ± 0.06 |
C | 100 | 5 | 1.51 ± 0.10 |
D | 50 | 5 | 1.23 ± 0.19 |
References
[1] A simple colorimetric method for viable bacteria detection based on cell counting Kit-8[J]. Analytical Methods, 2021, 13.
[2] Shengli, Pan, Yingying, et al. TRIM52 promotes colorectal cancer cell proliferation through the STAT3 signaling.[J]. Cancer Cell International, 2019.
[3] Roslev P, King G M . Application of a Tetrazolium Salt with a Water-Soluble Formazan as an Indicator of Viability in Respiring Bacteria[J]. Applied & Environmental Microbiology, 1993, 59(9):2891-6.
[4] Xiong Jianwen, Xiao Hua, Zhang Zhenxi. Comparison of test conditions for detecting cell activity by MTT and CCK-8 methods [J]. Acta Laser Biologica Sinica, 2007(05):559-562.
[5] Hou Chunmei, Li Xinying, Ye Weiliang, Cao Xiyuan, Xiao He, Li Yan. Comparison of MTT and CCK-8 methods for detecting cell proliferation in suspension [J]. Bulletin of the Academy of Military Medical Sciences, 2009, 33(04):400-401.