If You starve the cells before the wounding of the monolayer and use serum-starvation conditions after then there is no problem with cell growth and the decrease in the diameter of a wound during first 24 hours can be treated as the result of migration only. The wound healing assay analyzes the rate of a directed migration of groups of cells. It is not used to analyze migration of single cells, because in this assay You need to also remember about cell-cell and cell-ECM interactions (You wound the tight monolayer of cells attached to each other and to the plate) - it why it is directed migration - cells migrate only in the direction of an empty space (if You starve cells properly there is no proliferation, only migration). So You get quite a lot of information about the ability of Your cells to migrate, e.g., from the border of a tumour in the direction of blood vessel.

Usually, it is good to also check the ability of the tumour cells to invade ECM, e.g., using Boyden chamber with the filter coated with ECM, so You analyze the ability of the cells to invade ECM through dissolving of ECM using,e.g., metalloproteinases.

Together, both aspects: ability to migrate and ability to invade ECM can tell You if Your tumour cells really have invasive properties and can go from the main tumour mass in the direction of blood vessel and extravasate into bloodstream.

Ya it is done

It's done pretty frequently with cancer cell lines, especially in people studying aspects of cell motility and invasion. If you google your cancer of interest and cell motility, most likely you'll find some paper where they've performed a wound healing assay. Although there is some debate whether it is cell growth or motility. Shorter time points are best for wound healing in cancer lines.

If You starve the cells before the wounding of the monolayer and use serum-starvation conditions after then there is no problem with cell growth and the decrease in the diameter of a wound during first 24 hours can be treated as the result of migration only. The wound healing assay analyzes the rate of a directed migration of groups of cells. It is not used to analyze migration of single cells, because in this assay You need to also remember about cell-cell and cell-ECM interactions (You wound the tight monolayer of cells attached to each other and to the plate) - it why it is directed migration - cells migrate only in the direction of an empty space (if You starve cells properly there is no proliferation, only migration). So You get quite a lot of information about the ability of Your cells to migrate, e.g., from the border of a tumour in the direction of blood vessel.

Usually, it is good to also check the ability of the tumour cells to invade ECM, e.g., using Boyden chamber with the filter coated with ECM, so You analyze the ability of the cells to invade ECM through dissolving of ECM using,e.g., metalloproteinases.

Together, both aspects: ability to migrate and ability to invade ECM can tell You if Your tumour cells really have invasive properties and can go from the main tumour mass in the direction of blood vessel and extravasate into bloodstream.

Yes! I've done it! but you have to stop proliferation before, to avoid healing due to cell division.

Chandra Kishore, Elyse Walk, Patrycja Koszałka and Guillem Nadal thank you for your answers and valuable comments

So can any one provide me with the protocol please ?

Before the cells were seeded in a 96-well plate, on its underside the auxiliary lines (picture in an attachement showing one well of a plate with such lines, how You see it in a microscope - the one in comment below, because it did not attach properly here) were carefully drawn using marker with a fine tip (take a plate from a package, still closed put it on a table so You look at its underside and make a straight lines on a plate).

B16F10 cells were seeded in a 96-well plate (2x10'5 cells/well) and cultured until 90% confluent. Cells were subsequently serum-starved overnight, and a linear wound was applied to the monolayer using a 200-μl pipette tip. The line was done on the right or left side of the vertical auxillary line. Loose cells were washed away with PBS.

Images were captured immediately after wounding and again after 24 h. The wound width was calculated using arbitrary units with the use of ImageJ software (NIH): 5 straight lines were made across 'wounds' on a single photography, the length of the lines was shown in arbitrary units. The average values of them were calculated.

The cell migration distance was determined by subtracting the width of the

wound after 24 h from its initial width at time 0 (immediately after wounding) - it is why the auxillary lines on the bottom of the plate are needed - so You are able to find the same place on a plate and make a photo of it after 24 hours (photo 'd2 24 h' - auxillary lines in red). The values were plotted as the percentage of the wound closure, with the initial width set to 0%.

The first attachement did not attach, so here it is:

The scratch wound healing assay has been widely adapted and modified to study the effects of a variety of experimental conditions, for instance, gene knockdown or chemical exposure, on mammalian cell migration and proliferation. So it is practical to use cancer cells too.

Hi,

Thanks for raising the question and Patricja for your protocol. It seems my cells dont survive well when they are starved for 24. I decided to use 1% serum but again I see that they don go well with transfection and starvation. If anybody can tell how many percentage of serum will be an acceptable range for wound healing assay??

I have serum starved BxPC3 pancreatic cells for 24 hours then used 0.5% serum supplemented media on a scratch wound plate after scratching and they migrate well. We image in the Incucyte. 

However I am struggling to find any literature regarding the importance of serum starvation on stopping proliferation and the importance of serum concentration on cell migration vs proliferation. I require this for my students report and my thesis also. Can anyone direct me to such literature? Many thanks