So you have a gene or protein that you think may be involved in migration or invasion and the next step is to embark on migration assays. These assays are useful for testing fundamental migratory processes, such as embryonic development, immune response, metastasis and angiogenesis. For a long time these have been an invaluable mainstay of cancer research, however, gaining consistent results from such assays can be tricky, especially if handling 3D-matrices is not in your repertoire…yet! Here are some tips and tricks to ensure the best results from your migration assays.
Before You Start Anything
Everything starts with cells. Make sure to passage your cells regularly before these assays to keep them at 60–70% confluency before seeding.
Know how many cells to seed to make a confluent monolayer; usually a good concentration of cells to start plating is anything from 1 x 105/ml to 3 x 105/ml. Allow for cell growth during starvation and treatment beforehand if necessary. Starving cells in media containing 0.1% FBS synchronizes the cell cycle and depletes growth factors that could influence cell migration. GOLDEN NUGGET: serum starvation is crucial if you are adding enzyme inhibitors and want to see if they work specifically. Removing the serum helps you verify that it is the inhibitor and not the serum proteins.
Keep in mind that FBS contains a host of cytokines and factors that cells will migrate toward. When you study proteins that influence migration, the effect may be seen only when these factors are taken away, therefore, it is useful to titrate out FBS in media; usually 1% or 2% FBS is used for effect of growth factors on cell migration, but cells need to survive the assay!
For end-point migration assays, making a decision on the end-time point is important. Migration assays should be left for 24 hours or up to 48 hours. After that, the risk that proliferation could skew your results is high; however, this can be offset by a matched proliferation assay alongside your migration assay. Same rule applies for invasion assays but they are left between 48–72 hours depending on the cell line. If possible, measure wound width every 2 hours.
Mitomycin C is used widely to inhibit cell proliferation during these assays; however, the literature suggests this compound inhibits RNA and protein synthesis, so use with caution and at the lowest possible concentration.
Migration assays are straightforward in the sense that there is no matrix plating to worry about, but there are a couple of different techniques that will give you different answers by assessing different aspects of migration— whether it be a general change in movement/speed or the ability to sense and chemotax toward a signal.
This is an easy assay to set up if you have a time-lapse microscope or even better with the use of an IncuCyteTM and WoundMakerTM. Scratch assays assess the ability of a cell to move into a space (i.e. moving from a highly dense tumour to blood vessel or stroma). Cell lines that tend to form colonies as opposed to a monolayer may be a bit more difficult to assay, however, this may be remedied by coating culture dishes in poly-L-lysine so the cells adhere to the plate rather than each other. These kinds of assays can be carried out in a 6-well format right the way through to a 96-well format and for obtaining a straight wound without the use of a WoundMakerTM, simple tools such as a (sterile) ruler and pipette tip will work just fine to make straight lines.
After wounding cells it is a great idea to wash with serum-free media as many times as possible to ensure floating cells don’t settle in the wound and also to make your wounds as ‘cell free’ as possible. Overlay your cells gently with the concentration of media you have already optimized with or without conditions (inhibitors etc.). When using time-lapse microscopy, it’s a great idea to filter your media for a clearer image and to get rid of bubbles. Removing bubbles or “de-bubbling” (as we like to call it) is easily achieved by gently squeezing a wash bottle (containing 100% ethanol with the inner straw removed) to blow vapour over the surface of each well. Alternatively, you can set your pipette to a few microliters more than is needed and don’t depress the pipette the whole way, for example, if you need 50ul of media set your pipette to 54ul but keep this setting the same between experiments.
Chemotaxis assays allow you to assess a cells ability to move toward a certain compound, growth factor or bog standard media and can be bought as transwell assays. However, the main disadvantage is that there are no images of cells and there is only an end point (unless you are using a machine such as the xCELLigence or IncuCyte Chemotaxis module). Optimisation of this particular assay takes a little more time as you can’t see your cells and there are more handling steps involved. Beware if you are working with cytokines and signalling pathways, as use of common trypsin-EDTA will cleave off receptors on the cell membrane, which again will give unfitting results. Instead, use a more gentle dissociation reagent such as TrypLE®/Versene® (Life Technologies) or 10mM EDTA (pH8.0).
Invasion assays can be a scratch wound filled with EHS matrix or Collagen I/IV or a chemotaxis assay with a thin layer of plated matrix. Follow the same rules for optimisation as mentioned above to kick start consistent results!
Always allow for higher ‘dead’ volumes when plating 3D-matrices as they tend to stick to your pipette tips more. When plating, chilling the plate at 4°C for 5 minutes will help with consistent matrix plating (don’t worry, the cells can handle it!), and warming your plate to 37°C quickly ensures your cells will be happy. It is also an awesome idea to chill your tips, which will stop any premature setting of your matrices. Slight changes in 3D-matrix plating densities arise from common pipetting errors, which can mean inconsistent results especially if there are bubbles or the matrix hasn’t set completely before introduction of cells. Remember: bubbles cannot be removed when the matrix has set so take care when mixing and plating.
Usually 50% EHS matrix will take around 30 minutes at 37°C to set while 5% EHS matrix will take up to 4 hours at 37°C, other matrices will vary in their setting time. Always carry out a no EHS matrix control well as this will give you confidence in your matrix forming correctly.
Justus, CR, Leffler N, Ruiz-Echevarria, Yang, LV. In vitro Cell Migration and Invasion Assays.
Essen Bioscience. Live Cell Imaging in Your Incubator.
ACEA Biosciences, Inc. ACEA’s xCelligence DP Analyzer.