Is the 96- well cell scratch test so easy?
Shanghai Dianao Biotechnology Co., Ltd.
Cell scratch test: Classic does not mean no pain points
Cell migration and invasion are the basis for studying life functions such as tumor metastasis, embryo development, wound healing, and inflammation. However, the traditional approach is difficult to systematically compare the effects of different experimental conditions on cell migration. Although the scratch test is commonly used, it is difficult to carry out scientific and robust experimental analysis because the width, direction, and position of the scratch are difficult to maintain consistently , and the cumbersome operation causes the flux to be difficult to increase .
In addition, cell migration is a long-term dynamic process , but most of the current cell detection still uses the endpoint method , only the moment of an experiment, not the trend of the whole experiment.
So the question is, how can we make high-consistency, high-throughput, long-term dynamic scratch tests?
IncuCyte® Solutions
Figure 1: 3D cell invasion assay under the IncuCyte ® system lens
Invasive HT-1080 cells and non-invasive MCF-7 cells
In response to the above problems, Essen Corporation of the United States developed a cell migration & invasion application around the IncuCyte ® real-time live cell analysis system . Among them, the matching 96-hole scratching tool (WoundMaker TM ) can form 96 highly consistent scratches at the same time, greatly improving the robustness of the experimental results and eliminating the hard work of manual scratching.
The 96-hole scarper has 96 small scratched pins on the upper plate. Press the black button to move the upper plate to the side. At the same time as the upper plate moves, the scratched small needle will draw 96 equal width and straight scratches in each hole of the 96-well culture plate at the same time!
Figure 2: Comparison of manual scratches (upper right) and 96-hole scarifiers (bottom right)
After adding different experimental conditions to the 96-well plate, the IncuCyte® system built into the incubator was used to perform long-term real-time monitoring of the dynamic process of scratch healing. IncuCyte® accommodates up to six 96- and 384-well plates and quantifies the migration process for each well with powerful graphical analysis software . In the process of data collection, the cells do not need to leave the stable incubator environment, and the user does not need to repeatedly enter and exit the cell room.
Figure 3: IncuCyte® system built into the incubator (left) and real-time graph of 96-well scratch healing (right)
How important is long-lived live cell analysis?
The process of cell migration is divided into many steps and is regulated by a variety of signaling pathways . IncuCyte®'s high-throughput, high-consistency, long-term real-time detection allows users to simultaneously study the time-dose effects of multiple drugs. When Dr. Trezise's team inhibited cell migration (actin polymerization, mTOR, Hsp90, PI3-kinase) through four different pathways and performed a 40-hour real-time analysis with IncuCyte®, it was found that although all four methods can slow down scratches The efficiency of healing, but the time of maximum effect of each drug is different.
For example, the role of the PI3K inhibitor [ Wortmannin, lower right ] is most pronounced in the early phase , which is complementary to the PI3K signaling pathway that regulates cell migration in the early stages: cell polarity formation and extension of the pseudopod . The Hsp90 inhibitor [ CCT018159, lower left ] stopped the healing process of scratches in the later stage. If there is no long-term real-time observation, it is difficult to find this difference in time effect.
Figure 4: 40-hour real-time scratch healing observed by IncuCyte® for simultaneous study of time-dose effects of multiple drugs
(RWD% = relative density of cells in the scratch zone)
In addition to quantifying data, IncuCyte also preserves real-time image recording of the scratch healing process. We can also see that at the same concentration, Wortmannin and CCT018159 have different effects in time. In the early stage (t=6h), Wortmannin inhibited cell migration more strongly than CCT018159 (scratch is wider), but in the later stage (t=12h, t=24h), CCT018159 stopped the process of scratch healing.
In the same experiment, the result we saw at t=6h was opposite to t=24h . If we use the endpoint method to detect the results only at certain points in time, we can not only get comprehensive information, but also get different results because of the choice of time points.
Figure 5: Image results from t=0, 6h, 12h, 24h from the real-time record of scratch healing
Since IncuCyte can simultaneously analyze six microplates , we can screen a variety of factors that regulate cell migration, such as small molecules, biopharmaceuticals, and RNA interference. Dr. Trezise screened three different cell lines (3T3, HT1080) and added three different inhibitors to each of the three signaling pathways (PI3K, mTOR, Hsp90). The ability of 3T3 cells to migrate was found to be more sensitive to PI3K inhibitors.
Figure 6: High-throughput screening based on cell scratch assay
IncuCyte® System: Other Applications
Cell migration/invasion is a key event in physiological and pathological processes such as inflammation, wound healing and tumor development. Essen has developed scratch healing detection into a high-throughput, robust, simple, long-term dynamic image analysis method. Can be used for large-scale screening.
IncuCyte ® is an instrument that contributes more than 1,000 articles worldwide, and there are many applications, including immune cell killing, Chemotaxis chemotaxis, cell phagocytosis, etc. We will continue to analyze you next time.
Figure 7 Key applications of IncuCyte
IncuCyte S3 product introduction: http://?equipid=4153665&division=405
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reference:
O'Callaghan T, Alcantara LS, Dale TJ, Nelson T, Riggs AJ, Neagle B, Groppi V, Trezise DJ. (2011, September). High-fidelity_96 well_kinetic_imaging_assays_for_cell_migration . Poster session presented at the MipTec 20: European Conference and Exhibition for Drug Discovery at Basel, Switzerland.
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