Hey all
I want to image and analyze the movement of satellite cells on isolated fibers. My Setup is fixed everything works unless that I'm not able to immobilize my fibre in a way that it's not moving. For the moment after a couple of hours imaging, the fibre moves out of focus.
So far I tried to embed the fibres in Collagen. However, I have only access to commercially available Collagen 1 which concentration seems to be too low. Thus the acidic part is too high and cells start to die.
Has anyone experience in that? I was thinking about trying to do it in matrigel or agarose or a mixture of agarose and collagen.
Hi Denise,
We've used minuten pins into a layer of PDMS poured on the bottom of a culture well to immobilize the ends of fibers. You might be able to substitute dabs of modeling clay, Gorilla Glue (polyurethane) or something else a bit soft to hold the pins in place. The specific microscope objectives you use will *significantly* impact the tolerance you have to movement (depth of field). It could be a worthwhile investment.
Collagen is usually supplied concentrated at 3.1-5 mg/mL in dilute HCl or acetic acid to keep it soluble. Most protocols call for diluting it ~10-fold to enable gelling which of course raises the pH; if you still have pH problems, including a buffer such as HEPES should help. Cell culture grade low melting point agarose is also a great idea, although image quality will probably suffer. Maybe you could implant the myofiber on the surface of the agarose (with pins?), without embedding, to facilitate imaging.
I still remember time-lapse videos in a seminar by Terry Partridge many, many years ago showing satellite cells walking off of isolated myofibers while in culture. Quite spectacular. You should really contact him (or a protege) for the best advice.
Hi Denise,
Did you check this reference? Certainly the best movies of satellite cells on single myofibres.
Muscle satellite cell proliferation and association: new insights from myofiber time-lapse imaging
Ashley L Siegel,1,2 Paige K Kuhlmann,1,2 and DDW Cornelison
Skelet Muscle. 2011; 1: 7.
Good Luck
I didn't know this publication however they immobilize the fibres also in Collagen, this I already tried and it was not very satisfying. This is why I was looking for other ideas,
But thanks anyway!
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