Hello,

we are beginning to use Transwell culture plates to establish an air-liquid interface for culturing RPMI-2650, nasal epithelial cells (Bai et al. J. Pharm. Sci. 97: 1165-1178). The principle is to seed the cells in the upper chamber, let them attach for 24h, then aspirate the medium. The cells will receive all the nutrients they need from the lower chamber and they will differentiate and polarize. At the final stage the cell confluency should avoid any leakage from the lower chamber.

But we have no experience with the technique and some small tricky questions arise.

When we aspire the medium, we produce a scratch in the culture. We have tried with glass Pasteur pipette and with Gilson pipettes. The empty area is only very slowly occupied by this kind of cells. Is there any system for avoiding it? Either a better aspiration system or a different approach for emptying the chamber.

I will appreciate your help.

Thank you!

Hmm - how about aspirating the majority of the medium off, being careful not to touch the monolayer, then turning to some sterile, absorbent material (gauze, etc) to wick off the rest of the media?

Just my quick 2 cents.

Thanks, I was also thinking about something like that, but I am affraid it will be even more rude than the pipette tips. Although the extrem of the gauze is softer, I worry we cannot control the possible sweeping movements.

But for sure we are gonna test it. I will tell the result.

Any other idea/experience?

Thanks!

Dear friends,

I am having some problems with my transwell as well.
I never did transwell migration assay before, but right now my work in new lab requires me to validate previous experiment result regarding a gene's effect after transfected into the cell line.
I pre-treated my cells using mito C to stop proliferation, then seed the cells in transwell. After 22 hours of incubation (with Fibronectin as substrate and FBS as chemoattractant), cells that migrate to the lower chamber was counted. Problem is, the result is not very consistent.
According to the previous experiments, the expression of the gene suppose to aid in cell migration. Sometimes, I get less transfected cells migrate in wells with chemoattractant; sometimes, I get more transfected cells migrate in Fibronectin wells but not in FBS wells...

According to some of my labmates, it is very tricky (hard) to get consistent result using transwell. Not as easy as using scratch assay. Is there any tips and tricks in doing this?

Hi Sanjiun,

I do not get the sentence I have quoted. As I unterstand it, you have all wells coated with fibronectin and then some have FBS as chemoatractant in the lower chamber; and I assume some don't (as negative control). What do you mean you have wells with fibronectin and others with FBS?

For what I have heard, it is true that migration assays use to have a huge variability. If you have already selected the transfected cells and the expression efficacy is similar for all of them, I do not know what other factors you could influence.

But in any case I suggest you to repeat the question opening a new threat with a more specific titel. It will make it more visible and you may have more answers



That was my own question, and I think I have found the answer. Something quite simple: we take the Transwell insert out of the lower chamber, so we control much better the position of the Pasteur pipette and we can aspirate the liquid from the wall by capilarity. We have started the assays in that way, I will tell you if this is the final solution.

The next question is how to measure the viability of the cells. We are using the XTT protocol, but for doing the measurement we should trypsinize the cells, and up to now the efficacy is not very good. I will look in the forum if someone else asked the same question, if not I may open a new thread. Unles in the meantime someone answers here