I am working on a co-culture of two different cells using a polycarbonate membrane Transwell inserts to see if either one or both cells grow better together versus monoculture of the cells by examining their protein expressions using Alexa-Fluor 488 conjugated antibodies. The manufacturer did mention that polycarbonate membrane has a low cell visibility under a microscope. However I am hoping that I could make full use of the materials that I have in the laboratory right now. Are they any tips and tricks to best view the cells on a polycarbonate membrane Transwell inserts? Your advice is kindly appreciated.
Dear Nurul,
2% PFA is fine also for transwell polycarbonate membrane, we've performed several time fixation and immunofluorescence on this support. You must play particular attention for the focus, cause membrane and plastic are very close, since may be better to transfer the filter in another well in orther to better visualize the labeled cells.
Best luck
Dear Nurul
If you have access to a confocal microscope (CLSM) you may simply fix and permeabilise the cells, stain them with alexa-488 coupled phalloidin and propidium iodide, cut the inserts in small pieces and mount on coverslips with the cells facing the coverslip. This will allow you to see the cells, without interference from the polycarbonate support (due to the CLSM's ability to make optical slices)
BR Birger
Dear Dr Gargioli, did you mean that you view the cells while they are still in a well-plate?
Dear Dr Brodin, cutting the inserts in small pieces sound like a very good suggestion! I will definitely try that for my experiment. Thanks!
Hi Nurul,
yes we perform immunofluorescence on the dish and then we llok at the results by inverted fluorescent microscope!!!
Good luck
Nurul Adhwa Rahman - I came across this post and wondering if you were able to image your cells properly without interference from polycarbonate membrane? I need to do this too during next month. Thanks!
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