We are observing some chemotherapeutic drug effect in Boyden chamber migration assay using H460 lung cancer cell line and the following standard procedure. But we didn't observe any movement of cells across polycarbonate membrane. Can cells number increases give some effects? or should the polycarbonate membrane be treated with something first? Any suggestion that can resolve problem?
Coating the PET membrane with extracellular matrix proteins will test the invasive ability of H460 lung cancer cells. Incubation time, as well as number of cells is critical.
Regards
Alaaeldin
I agree with Alaaeldin, if you coat the membrane with any ECM it tests the invasive/degradative abilities of the cells, not just migration. And also number of cells and assay time is important. You may need to do some optimization to find the right number of cells and amount of experiment time. I would also play around with the stimulus you add to get the cells to move. A lot of the time just using FBS works, but occasionally you'll have to add more of a specific growth factor. You should also be sure your cell size and membrane pores are compatible as well, as that could be hindering movement.
My troubleshooting questions would be 1) have you coated your membrane with ECM? what type? what is the pore size on the membrane used? how long you left your cells to migrate? have you used a negative control and a positive control. i.e. PDGF to ensure that the experiment actually works? are you sure you did not wipe out the face where the migrating cells are? Have you fixed your cells and stained them before counting?
I have worked with human airway smooth muscle cell (HASMC) migration toward different stimuli, I noticed that cells don't adhere or migrate when I don't coat my membranes ( I usually use 0.01% collagen I rat tail). Sometimes using high concentrations of ECM may hinder the cell migration. Using both negative control (medium alone) and positive control like PDGF-BB (10ng/ml) is essential in each experiment you run. It is advised to use 1 million cells/ml, but in occasions where I had limited number of cells I used less. The results were reproducible as long as I load the same number of cells in each well in a given experiment, since I calculate the fold increase compared to the negative control. For HASMCs migartion, 4-6hrs incubation time using 8-10uM pore size was sufficient to see difference in migration. Most importantly, once you optimize your conditions stick to them for better reproducibility.
I hope this helps.
Good luck,
Laila
It depends what you want to test for? Testing for cell motility, there is no need for adding collagen (or matrigel). If testing for cell invasion, adding a matrix like collagen will be needed. Adding collagen will require the cells to both migrate and invade (remodeling the collagen)
If you are specifically looking at migration and not invasion/matrix degradation, boyden chamber assays aren't great. Even with a thick matrix coating, it is difficult to maintain a chemotactic gradient over a long period of time. Without one you lose the gradient in less than an hour. Have you looked at Dunn chamber assays? Or wound closure?
Thank you so much every respondent . Your immediate reply is very helpful.
@Laila Al-Alwan: No i didn't coat with any ECM because i think that it is useful only for invasion. but i want to test some chemotactic movement after drug treatment. second pore size is 8uM. and incubation time is between 4 to 6 hrs. yes fixed in methanol and then stain with wright stain. your comprehensive reply i appreciable.
@Jessica Neisen, thank you for your suggestion. but i read some papers used same cell lines and boyden chamber and they got comparable good results thatswhy i select boyden chamber assay.
You're right, Boyden chambers are standard practice for migration. From my experience though, If you actually check the chemotactic gradient using a chemokine or growth factor that you can quantify by simple ELISA, run samples from the top and bottom chambers collected over a period of time, and you'll see that the concentrations equilibrate within an hour or so.
This isn't a problem if you expect your cells to respond to treatment immediately and start moving. Boyden chamber assays will work just fine. However, if the cell response is more complex, i.e. drug needs to be metabolised or gene expression needs to be switched on/off, your motility response may not occur until after you've already lost your chemtactic gradient.
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