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

Hello experts,

I am doing some tissue culture work with cancer cells and was wondering if anyone knew of any quick and simple methods to measure cell motility or invasiveness? A lot of the assays for cell motility seem too time consuming. As a last resort, I suppose there are kits out there that serve this purpose. If any one has had any experience with them please let me know! Also, would anyone happen to know of any tissue culture assays for identifying tumor suppressors or oncogenes? Thanks for your time!

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Hi!

I 've studied invasive activity of different primary tumor cells and testing anti invasive activity of some substances. with Matrigel invasion assay. well this assay expensive but it gives more reliable data in most cases. If you want to do important experiment to prove your suggestions in your work use it may be on the final step. but if you want to do pilot estimative experiment not to deepen use the following: wound healing assay to estimate cell motility and may be gold particle assay.

Wound healing assay . Seed your cells into 24 well culture plates and culture till tight monolayer will be. than take a tip and make a straight line simulation "wound ". Then wash your cells with appropriate culture medium several times to remove cell debris. Make it in three parallels each. Don't forget about negative and positive controls. Then after several day ( it depends on your cells) cells from the edges migrate to each other from opposite sites. Semi-preicise estimatation you make by checking thickness of your "wound" during time. Before making exp you should spend your time on analysing time period till your cells remove wound ( close your wound line) without any substances. Training a little with making line with tip. It is not difficult only press tightly your tip to bottoms of plate. I did this assay it with HT1080. Work good!

Another assay deal with observe tracking og your cells in colloidal gold( it is not expensive subs from Sigma) to test cell motility.

Concerning invasion assay I think that you can't find cheap assay. and you see that this assay demand some skills ( if you will prepare cell inserts with matrigel but not buy ready to use inserts. So this work will caught not only yuor money but a time to adapt to this method. I recommend your to buy a book from Humana Press called Metastasis research protocols in series Molecular medicine. In first volume given a lot of info about your task.

Good Luck!

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in addition to circlepoint: culturing cells in Transwell plates; membrane size between 3 -8 µm for migrating cells to enter the lower chamber

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o the wound healing assay seems like a good idea. I'll look into that. But, how do you calculate the rate of migration? Counting the cell # at different time points? And, the bearer the 3 -8 µm membrane size in reference to another assay, not the wound healing right? Are you refering to the cell migration assay?

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yes, a different assay: you use two chamber multiwell culture dishes: Cells are cultured in the upper chamber on membranes with pores, and may migrate through these pores into the second chamber; you may count these invaded cells or stain total protein after fixation; Transwell plates are not cheap but apply for a sample (NOT Millipore, they want money for a sample (!) (50% of normal price) , better try Cornwell)

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I' ve estimated the rate of migration by making a foto during experiment ( for my culture 1, 2, 3 days) under the same microscope and camera settings. Then you can estimate wound thickness of each image. The rate of migration activity is:


% = (thickness (when you start exp) - thickness ( on the other day) / thickness (when you start exp))*100

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Various cells have different migrate patterns. I mean one kind of cells ( most of all which grow in suspension) may migrate in low compartment area and others typically attach on the other side of insert membrane. In my work I've used BD cell culture inserts. Pore size choice 8-12mkm depends on size of your cells. 8mkm is most popular!

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circlepoint, this sounds very interesting but how do you measure thickness of a layer with a microscope; do you use a CLSM for a xyz-scan? and for it, don´t you need a stain?

moreover, if you have a cell line which only grows in monolayer, is measuring thickness useful?

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Well! In this assay It is not need to estimate thickness of cell layer. When I speak about thickness I mean width of "wound" . Excuse for mislead

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hello again,

sorry for the simple question but its still not that apparent to me how to measure the "thickness" of the wound? you take a picture and measure the wound thickness by ruler?

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