Quantifying invasion assay results - Light microscopy as alternative to colorimetric (Nov/29/2007 )
I've been doing invasion assays and quantifying the results via colorimetry, but am getting lots of noise from nonspecific staining of the dye that doesn't completely rinse off. I'd like to try counting via light microscopy, but have not done so before and would appreciate some help.
Do I need to detach the filters on the bottom of the Boyden Chambers in order to count, or is there some way to leave the entire setup attached?
How do I choose which fields to count, since invasion is visibly not uniform across the entire bottom of the well?
I currently have an inverted phase microscope and a standard microscope to work with in the lab. We have no digital imaging software or equipment to work with. Any advise in addition to answering these questions would be appreciated.
-TheSquire
Do I need to detach the filters on the bottom of the Boyden Chambers in order to count, or is there some way to leave the entire setup attached?
How do I choose which fields to count, since invasion is visibly not uniform across the entire bottom of the well?
I currently have an inverted phase microscope and a standard microscope to work with in the lab. We have no digital imaging software or equipment to work with. Any advise in addition to answering these questions would be appreciated.
-TheSquire
we do not know exactly what you like to do;
there are stains such as trypan blue where you can simply discriminate between stained and non-stained cells; so counting is easy; if your clometric stain is the result of a physiological reaction, it will be difficult to discriminate different intensities just by microscopic inspection without software support
The stain is the one that comes with the kit, which I think is a crystal violet stain.
New question/problem:
When I look through the microscope at the membrane after having mounted it on a slide, I see a lot of places where there looks to be a cell on/in the pore itself, except that in most cases it appears at 200x that only the edges of the pore have picked up stain and not the area over the center of the pore. At 400x it looks like there might actually be a cell over the pore. Are these actual cells, or is this just an artifact of mounting the membrane on a slide? I ask because I'm seeing this even in areas on membranes where there are little to no areas of staining visible to the naked eye. The patterns of staining visible to the eye roughly follow what is reported in the literature, but if all these dark-rimmed pores are actually invaded cells then there is no difference in invasion between cells exposed to vastly different amounts of the compounds I'm testing, which makes no sense.