Cell motility/Invasiveness assays - (Mar/07/2007 )

yes - it is somehow tidious as the line of wound is not uniform;

for more advances in wound healing and cell migration, take a look at Keese et al., PNAS 101, 1554-1559 (2004)

http://www.ncbi.nlm.nih.gov/entrez/query.f...l=pubmed_docsum

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Thanks The Bearer here is interesting approach for estimation cell migration. But sometimes as procedure cheaper and easier that is in inverse relation to accuracy and significancy, mainly but not always. If we will use measurements of current and grow cells on electrodes so we can increase accuracy of experiment, but make it more expensive. And there is other problem, about influence of electric field on cell behaviour of cells ( for ex. migration to each other ). So this component like another "X" in equation.

Because in this topic we discuss cheap and not sophisticated methods so we should take in account borders of this assay. The main aim of wound healing assay is more qualitative than quantitave estimation potency of your compound on cell migration activity. You can make it semi - quantitave with help of ruler or photoshop tools with image on PC. If you have areas of wound with different width so measure for ex. 5 random areas along your wound with different width and calculate average. If your exp. substance calculated width differ from width of negative control less or equal 10% , so you substance seems show a little action on migration activity of your cells.

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you´re absolutely right; and that is what I meant with "more advances" assaying beyond simple scratch wounding; we mainly use wound healing assays for transformed cells; but if transformation affects cell-matrix or cell-cell contacts, one get different results of lined woundings which makes it difficult to interpete healing;

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Mind telling more about the measurement? U see, i managed to measure the gap of the 'wound' before and after treatment. However, I do not know how to present my data. I am thinking or calculating the rate of migration ( in %) within the duration of 8 hrs. Do u know how to calculate the % or migration? I was thinking of doing it like this:

(wound size Before treatment)-(wound size After treatment) x100%
(wound size Before treatment)

is this correct?

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The Bearer,
I am about to start some migration assays, using Boyden chambers. I am using adherent cells and my idea is to incubate the cells for 6-24hrs, then fix and stain with DAPI to count them. The problem is that here none has done such an assay before, so I have only hints from papers, where usually no complete protocol is described. I have few questions for you and the other experts, it would be great if you could help me.
1) after migration has occured, should i rub the upper surface of the filter with cotton swab? Is it enough to remove the non-migrated cells?
2) can I then fix the cells with paraformaldeyde or should i use another fixative?
3) how do you stain the cells? do you remove the filter to stain them?
4) do you mount the filter on a slide? is it possible instead of removing and mounting the filter, to detach the migrated cells by immersing the upper chamber in trypsine and thus let the cells fall in the lower chamber, so that it is possible to visualise them by use of an inverted microscope?

Thanks in advance for your help, looking forward to receiving your replies!

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Hi,

In addition to these methods, you could also use the inverse invasion assay. Similar to the one the bearer suggested. But you add the cells on the bottom of the transwell and let them attach before putting the transwell into medium. The transwell should be filled with matrigel or such and on top of that a chemoattractant. Stain with whatever-fluorescent label to trace the cells and analyse in a confocal microscope...

DLY

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I'm looking into the wound healing assay too. I'd like to know what would be a suitable serum percentage in the media that i should work with for this assay as i'vve read some where that if the percenate of serum is too high it may induce proliferation rather that cell migration. And besides that, what can i use for positive and negative control in this assay? Can someone help me?

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I guess your control should take care of proliferation problem, but I also guess you may keep the serum low (2%), or very low(0). Afterall, the assay lasts 24 hours, cells should not suffer much! Depends upon your cells too.

Positive Negative depends upon your particular expt. For example, if you have stable clones with a gene X in cell line Y, and you are looking for migration defect, you may want to also make a stable clone for a gene known to do so in these Y cells. Better request such clones from published labs.

Check some of these links :
http://search.vadlo.com/b/q?sn=158621799&a...assay&rel=0
..

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For the scratch test (woundhealing), you can use Mitomycin C to inhibit proliferation, if the assay goes for 16-24 h. For NIH 3T3 fibroblasts, I let them migrate for 16 h. For mouse embryonic fibroblasts, 16 h is too long and they will close the wound, so I let them go for 6 h. Mitomycin C can be bought from Sigma, beware that it is not very stable.

ETA: I use 5% or 10% serum. The migration will be reduced or stopped if you use very low serum because they need to growth factors to move.

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