It's a possibility that you can't rule out completely. Usually, the best is to perform the migration/invasion experiments within a short time, that is, below doubling time of the cells. You can manage to do that, setting up the conditions of the invasion / migration experiments (e.g. matrigel concetration, initial number of cells, etc..). Of course, this will depend on the invasion/migration capabilities of your cells. I'd say that you should be able to perform an invasion/migration experiments in 6-8h interval.

Good luck!

Do you know how this miRNA affects the proliferation? Is it through apoptosis? In that case you could try to study the effect of your miRNA on invasion/migration in presence of apoptosis inhibitors.

Hi Giacomo, thanks I was actually planning on analyzing the effect of the miRNA on apoptosis next week once I have my cells grown back up. Thanks for the advice. Once I have the apoptosis result, I will know more. Do you have any suggestions on how to perform the apoptosis assay?

In my experience the best apoptosis assay is AnnexinV/PI staining and FACs analysis...but there are many commercial kits available which you may use. You may also want to check if the apoptosis is through caspase-3/7, there are specific inhibitors that you could use to analyse the invasion/migration. Good luck!

It's a possibility that you can't rule out completely. Usually, the best is to perform the migration/invasion experiments within a short time, that is, below doubling time of the cells. You can manage to do that, setting up the conditions of the invasion / migration experiments (e.g. matrigel concetration, initial number of cells, etc..). Of course, this will depend on the invasion/migration capabilities of your cells. I'd say that you should be able to perform an invasion/migration experiments in 6-8h interval.

Good luck!

Hi Keith, you could stop proliferation with mitomycin C. I usually treat my cells (keratinocytes) 2 hours with 10ug/ml mitomycin C before plating them into the transwells. This will stop proliferation for good but your cells will still be alive for 48-72 hours and be able to migrate. One thing you have to consider is that you can't use mitomycin C if you are working with p53 as it changes its status.

You could do a proliferation assay (MTT or XTT) on your cells to ckeck the effect on proliferation.

I agree with Miguel, a short time period in the migration assay is an easy way to minimize potential proliferation or apoptosis effects. In cell lines with decent mobility, even 4 h should be fully sufficient for transwell migration assays.

How about count the cell numbers under the same conditions and time scales. Maybe a simple XTT assay can also answer the question.

Hi there Keith,

This is something our group constantly battle with!

It's hard to mimic the situation your cells are in during a migration/invasion assay (3D I assume, in a matrix), and therefore hard to mimic this in a proliferation/growth/apoptosis assay.

It's not a perfect mimic of cells invading in matrix but what we have done is perform basic SRB assays in parallel with the invasion assay.

If over the 24-hr period of the assay we see no effect on proliferation (SRB), then we're confident the effect is specifically anti-invasion.

You may, over longer periods of treatment, observe inc apoptosis, but I guess from a therapeutic perspective, that's a positive thing, as you are affecting >1 hallmark!

Best of luck,

Dave

Hi Giacomo,

If you perform migration assay (4-6h) using 24h serum starved cells, you can eliminate the effects due to proliferation. However, to determine the role of apoptosis, you should check the viability of cells and/or use PI staining to ascertain the membrane integrity to confirm whether miRNA induces apoptosis or not.

Good Luck

For migration assay, you could try time lapse imaging with specific chambers (eg, Dunn chamber, Ibidi chambers etc). So, you only measure a displacement and then you could observe a defect in speed, directionnality and formation of migratory structures such as lamellipodia. This is, in my opinion, the best way to get rid of any proliferation problem.

Just assess for proliferation with any technique accessible - MTT, thymidine, BrDU etc.

BrDU will be good since you can stain the cells with the antibody and get a visual too.

As others have mentioned, I always do a viability assay over a few days to assess whether my variable is causing any significant positive or negative effect on cell number before I try migration/invasion assays.

Hopefully over the 1-2 days of your migration/invasion assays you wont see any change.

Good luck

I am doing scratch assay for migration and I think using serum free or very low serum as mentioned in of the papers you cited also works. One way to make sure especially in monolayer culture is to see if cells are multiplying on top of each other.

It depends on your assay. Mitomycin C treatment is a gold standard for "wound" or "3D" assays as it will stop mitosis but not migration. However, I prefer Boyden chambers assays. An epithelial cell requires major cytoskeleton changes to go through the small pores not associated with cell division. Also include a non-stimulated control

The cell migration assays that I run are only about 3 hours, which is too short to have a change in proliferation. For both invasion and migration assays, I have also plated a separate plate with the same conditons and on the same ECM without the transwell insert and counted the cells after the assay. This helps control for both proliferation and adhesion issues. Also, as stated previously, timelapse is also a good way to watch cell move and get displacement and persistence parameters, but you can also see if cells are dying or proliferating more quickly.

If you have access to the equipment to my opinion you can get the most convincing data about migratory behaviour by live cell imaging under serum-free or low serum conditions. If you think that your miRNA has secondary effects, as additional controls you can add specific inhibitors or activators, if available and depending on what you know in this respect. In addition you can label e.g. the actin cytoskeleton and check if your miRNA induces changes in the actin cytoskeleton typically seen in migratory cells. Analysis of invasion in matrigel or other 3D conditions is more sophisticated.

Hi

you can treat your cells with MItomycin C or Hydroxiurea. Both are able to block cell proliferation keeping migration properties. You can do wound healing assay to verify the effects of your miRNA on migration avoiding cell proliferation using one of these compounds. Good Luck :)

Hi,

We developed an assay in the lab that enables to visualize the tracks of individual cells following a short 4 hr incubation time with drugs . The phagotraker assay. It is an assay suitable for screening drugs that affect cell motility.

I agree with all commentaries ; I do like said Miguel Segura above. Yael Sagi suggests the phagokinetic track assay (I published years ago papers with this technique). Rather it relates migration onto basement membrane compounds (Hapotactic migration) which is different from invasion assay in ThinCert (Boyden chamber) through matigel that requires both actve metalloproteinaes and chemotaxis : it is proper a invasion test.

I asked a question about anchorage dependent-clonogenic assay, but got no answer ! Is anyone able to help me ?

You can also kill two birds with one stone by looking at mitotic figures(radioisotope technique) of cells plated on a substrate containing a uniform layer of a nanoparticles(I used nanogold in a fibroblast migration assay I did last summer using Scanning electron microscopy). Its more like a sandbox which leaves tracks that shows cell migration(if any), following which you can score your mitotic index. Good luck. I can email you some papers if you are interested.

Hi,

I agree with comments of Miguel Segura and Ramesh Ray. The incubation time for invasion/migration assays are usually shorter than doubling time of the cells.

In my experience, when I test anti-invasion/migration effect of natural products, I perform cytotoxic and cell growth assay (MTT assay) in 96-well plates, at the same concentrations of test compound in similar condition. I usually perform all the assays in completed media (10% FBS) and incubate 24 h (for invasion/migration/cytotoxic assays) and 72 h for cell growth assay. The cells are allowed to proliferate in all experimental conditions, but invasion/migration/cytotoxicity are determined at the time that shorter than proliferation is measured.

If MTT results at 24 h (cytotoxic assay) show that cell viability >90%, while invasion/migration rate are significantly decreased, you can say that the inhibition of invasion/migration by the test compound (or miRNA) are not due to cytotoxic effect. In other words, the test compound (or miRNA) does not affect healthy of the treated cells during invasion/migration process occur.

Although the test compound (or miRNA) may decrease cell growth after 72 h incubation, but this effect occur later and may or may not involve the mechanism of anti-invasion/migration of the test compound (or miRNA). These experimental plan can simply differentiate anti-invasion/migration effects of test compound from its anti-proliferation effect.

If your cancer cells are highly invasiveness, you can reduce the incubation time for invasion/migration/cytotoxic assays to 6 or 12 h, while keep 72 h for cell growth assay, these will improve the differentiation between anti-invasion/migration and anti-proliferation effects.

Good luck!

I think you have to do 3 experiment to confirm 3 observation.

1. At 1st u should know the generation time of ur cell line, if it is less than 24h then for cytotoxic assay ,u can do by MTT. Here u should plate same no. of cells at a time for 24h, 48h and 72h (or more time according to generation time of cell line) and simultaneously another set like the same time point as before for cell counting by haemocytometer. This is the double conformation of cytotoxic assay.

2. for migration inhibition u can do the" Scratch Assays" after treatment with mi-RNA.

3. For invasion as u did previously by "invasion on ECM coated transwell inserts"

May the these 3 techniques will help u.

Good luck.