So previously i grew cells as a confluent monolayer on transwell insert with pore sizes that prevents migration. Then once the cells are attached, i place a 5mm thick agarose-dmem agar cushion over it followed by compression cup with a certain weight to press down on the cells. I did immunofluorescent staining on this but image wise even with confocal i was having difficulty with getting clear image. So i was thinking of lowering down the complexity of my design.
I intend to just seed the cells on coverslips in a 35mm plate. Once attached, i place an agarose-dmem cushion over it, then the compression cup. The placement of the agarose cushion and cup is to be done 24 hours after seeding and when media has been aspirated out to allow easier placement of these 2 components. After the placement, i will add media to the sides slowly.
Here are potential problems i forsee. Please highlight more if you can see any.
1) if i cant place the agarose cushion in 1 vertical motion that would the surface of coverslip such that i have to make sweeping horizontal adjustments of agarose cushion, i might dislodge the cells or affect the morphology of cell. This might complicate the after compression morphology i want to see.
2) similarly, during removal of agarose cushion, it has to be done carefully such as there is no sweeping motions. Maybe this has to be done when liquid media has already been aspirated out?
3) will compression itself onto cells attached to coverslips dislodge the cells? I can imagine that adhering onto coverslips is weak.
I think the answers to the question you have depends on your exact skills and to answer them you need to perform an experiment. The changes in cell morphology may be observed by conventional light microscopy if you take the coverslips with grid to track the cell morphology before and after the cushion placement. The exact cell adherence depends on the cell line and the coverslip brand, so you may test to find the best combination for your cells.
What was the time you incubated cells under pressure? The cultural inserts could prevent the cells from being hypoxigenated in the case of long-time incubations.
I am currently trying it on coverslips. Yes i do worry if hypoxia will be an issue without transwell inserts. I am doing compression for 16 hours.
I place cells adhere on coverslip on 35 mm plate, a 24mm wide and 4mm thick circular agarose-DMEM cushion over it and then a compression cup that has same size as cushion. and then i slowly drop down 2 ml of media by the sides. do you think media can 'seep' into cells sideways? if not, 16 hours of incubation is hypoxia condition?
Yes, 16 h may be hypoxic conditions. You may test it reliably by western blot on HIF-1a (after removing the agarose cushion scratch the cells from coverslip to nearly 0.5 ml of RIPA buffer and proceed to Western) - like in http://www.jove.com/video/2899/induction-and-testing-of-hypoxia-in-cell-culture
You may also quickly observe if you have a robust hypoxia by staining the nuclei of your cells and observing apoptotic fragmentation or picknosis (for example, with DAPI or Hoechst). It is a very rapid procedure and you may do it along with immunocytochemistry for your proteins of interest.
It is also intersting to consider membrane culture dish (like Lumox, Sarstedt), when you can perform confocal microscopy directly on the dish bottom. Membrane will ensure your cells are normoxic and you may watch your cells even in vivo without cushion removal (but without immunofluorescent staining - you should use fluorescent fusions with your proteins of interest then). And maybe the membrane with cells may be fixed and stained as coverslip.
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