Invasion assay, preincubation, and confounding - What am I really measuring? (Oct/17/2007 )

I'm testing compounds on a cancer cell line to see if/when they stop invasion through an ECM. The procedure I'm using, which appears to be standard, is to grow the cells to near-confluence, preincubate for 24hrs or so with the compound, detach and place in the upper chamber in serum-free media, with the lower chamber containing media with serum.

My issue is that, while I get inhibition, I'm not sure if that's mere inhibition of invasion, or inhibition of cell attachment/adhesion. Is there a test I can run that determine if I'm inhibiting attachment/adhesion?

Hi,

A good way to see if you're inhibiting adhesion is to do a simple adhesion assay. Grow the cells as before, trypsinize and count, plate out at set concentration and then in non chambered dishes count the number of attached cells after a certain time point (it will depend on your cells). You can fix them and stain with something like crystal violet or H&E stain to make them stand out more. That should give you an idea as to weather the adhesion is being affected or not.
There are other modifications you could try to your original experiment to see different parameters I'm sure.

Anyway good luck

as always
Lost in the Lab

My issue is that, while I get inhibition, I'm not sure if that's mere inhibition of invasion, or inhibition of cell attachment/adhesion.




The inhibition of attachment is part of the inhibition of invasion. You could of course run an adhesion assay, a toxicity assay AND an assay to determine doubling time of your cells for example but then you would have to test every compound several times.

This invasion assay that you perform seems to be a sort of screening assay to me. That is in a first attempt to find out which of your compounds is of further interest you run this invasion assay and see if invasion is inhibited. Basically you dont care why the invasion decreases you just find out which compounds have that effect. The inhibition you measure is the sum of several parameters (attachment, vitality,...).

Than in the next step you could do an attechment assay like lost in a lab proposed or other assays of your choice with the inhibiting compounds.

Thanks, coastal and lost, for your insights. I'll look into various adhesion assays now. Invasion assays are really expensive, so I'm reluctant to try lots of variation on that theme.

Hello The Squire!

Sorry for belated reply, another way to solve your problems is to incubate cells with your test compounds on ECM matrix directly in upper chamber, then wash several times with serum free media , add serum containing media in the lower well and follow invasion assay. I did some experiments in such a manner.

Circlepoint: I take it that, since there was no media in the lower chamber, you had no issues with premature invasion?

The Bearer: I see invasion in 24hrs with the cells I'm using, some people use 18hrs.