do you see any changes in actin staining patterns. I did mechanical compression on MDA-MB-231 cells. basically this involves growing cells as a monolayer on transwell insert, followed by placing agarose cushion and then a weight on it.
top image: compressed actin (phallodin red)
bottom image: uncompressed actin (phallodin red)
Dear Iwan,
I guess this a images of an normal fluorescence microscope? Do you have the possibility to acquire the images on a Confocal and do a z-stack? I guess without that it is difficult to answer your question about the staining pattern.
hi lukas,
does this explain why the staining is not clear? yes im doing immunofluorescence on transwell insert membrane. the membrane after mounting is not equally flat on the glass slide, thus i cannot get focus for every single cell in the image (few cells will be in focus, while the rest are not, sort of thing)
Based on the images it is not really possible to give a clear answer due to the different focus. If there is no change to do images on a confocal you might have the chance to use a microscope of Zeiss with an Apotome, or this is not avaivable too you can acquire images in different layers and use software to sharpen them to one (I have too look up the name of the ImageJ Plugin).
In the simplest way use a lower magnification which decrease the deepth of focus.
Hi Iwan,
No answers, only questions so far.
What are the images? With and without weight? Are both of them covered with the agarose cushion?
The compressed consist of DMEM-agarose cushion and cup with weight on it
The uncompressed only consist of DMEM-agarose cusion.
Am i really not seeing actin changes or is there a fault to my experimental design
Hi Iwan,
I m not sure your image show a monolayer of cells which I suppose are expected to be all adherent to eachother in that case. Therefore, beside acquiring z stacks using confocal, that for sure will provide better imaging of the actin in cell, might be usefull to have your cells in culture growing for really higher density to reach a monolayer if possible ?
Have you tried changing the concentration of the agarose and compared a low percentage gel (0.5%) and high (3%)? The gel stiffness will be an important factor, often leading to a cellular shape change by itself (see Laevsky and Knecht 2002 J Cell Sci). Also if you are compressing with a weighted cup the compression force needs to be equally distributed over the gel surface. Otherwise the gel could be compressed to varying degrees. Using a circular coverslip between the weight and the gel should even out the compressive forces across the surface.
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