High-throughput cell migration screening
Cell migration is a key process that is indispensable for living organisms to grow and sustain life. In order to perform the corresponding functions, cells in the body often migrate to specific locations in a specific direction. Migration is a cyclic process in which the cell protrudes from the front end and retracts to its tail. The migration of cells in animal tissues is primarily in response to specific external signals.
Cell migration is a very important process for embryonic development, wound healing, differentiation, and immune response. Cell migration is the pathogenesis of diseases such as vascular diseases, chronic inflammatory diseases, and tumor formation. Therefore, studies on compounds that promote cell migration (invasion of wounds) or inhibition (tumor formation) have very important therapeutic implications. We have established a new, automated method for high-throughput cell migration analysis that is standardized using Molecular Devices ' SpectraMax paradigm multi-detection platform. This analysis method is simpler and more reproducible than scratch analysis or other 2D wound closure experiments.
Test description
Oris cell non-coated ( Plattypus Technologies , Madison , WI ) used cell seeding stoppers to form a 2 mm diameter detection zone in the center of each well . Human melanoma cells ( 50,000 per well) were planted into 96- well plates fitted with cell culture inhibit plates. After overnight culture, the cells were attached to the bottom of the microplate, and under the action of the suppression plate, a central blank area of 2 mm was formed . The plate was removed and the cells were either left untreated or treated in a concentration-dependent manner using the soluble chemokine CXCL9 , or treated with 100 ng/ml of different biologically active compounds or latrunculin ( 1 ug ) as a negative control. Then, the melanoma cells were further cultured for 16 hours. Using CellTracker Green (Invitrogen) cells were stained, the bottom microtiter plate was added Oris detection mask (Detection Mask) (FIG. 1). Cells that migrated into the detection zone were imaged using a confocal microscope ( Leica TCS SP2 ) and the data was quantified using the SpectraMax paradigm assay platform (Figure 2 ).
Figure 1 : Principle of Oris Cell Migration Test
Figure 2 : Paradigm Detection Platform
Figure 3 : Screening for potential cell migration stimuli or inhibitors. The extent of melanoma cell migration was measured by reading the fluorescence intensity of Oris cell migration (with a detection mask attached to the microplate bottom ) using the SpectraMax Paradigm detection platform . The test was done with three duplicate holes.
result
To investigate and evaluate the migration effects of newly isolated active compounds, we used human melanoma cells treated with different potential migration stimuli or inhibitors (chemokines CXCL9 , Latrunculin and active compounds). We assessed the extent to which cells migrated into the detection zone by measuring the fluorescence intensity after 16 hours (endpoint assay). Figure 3 summarizes the screening results for melanoma cell migration effects of compounds. CXCL9 showed an increase in fluorescence intensity in the dose-dependent detection region (migrating cells), whereas Latrunculin had no migration effect. Although many active compounds did not show or showed only a very low migration effect, compounds 12 and 15 significantly promoted the migration of melanoma cells into the detection zone. The method of calculating the doubling induction level: the fluorescence intensity value ( RFU ) of each well divided by the average RFU value of the negative control . The average and SD values ​​of each sample were also calculated.
in conclusion
This experimental system confirms a useful tool for high-throughput cell migration and high-throughput drug screening. This study combined Oris cell migration assays with the SpectraMax Paradigm assay platform to quantify the extent of cell migration by measuring fluorescence intensity. This system is capable of distinguishing a wide variety of cell migration stimulators and inhibitors.
Author:
Christoph Wiesner , Maren Pfluger , Constantin Bujnow , Katrin Eisenberger , Wolfgang Schutt , Andreas Eger and Harald Hundsberger
contact:
University of Applied Science Krems Krems , Austria
Harald.
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