Author Topic: Single-cell suspension from dermal tissue  (Read 5909 times)

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Offline rockchalk

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Single-cell suspension from dermal tissue
« on: July 15, 2008, 06:24:16 PM »
I am attempting to take murine dermal tissue from an adult mouse and convert it to a single-cell suspension for use in FACS.  I am using a combination of collagenase IV (0.2%), dispase II (0.2%) and a touch of trypsin (0.05%).  I am hoping to get RNA out the back end of the FACS so time is of the essence.

Does anybody have any experience with this type of thing?  After 1 hr at 37C I can pass the slurry through a 70 um cell strainer (although not easily) but there is still clumps of debris present after resuspending my wash.  Should I place the tissue back into my enzyme bath after passing through the filter for a bit longer to dissociate the remaining chunks?  I need to have an accurate cell count on a hemacytometer in order to incubate with antibodies.

This is all very new to me and any advice would be greatly appreciated.

Thanks,
Stephen

Single-cell suspension from dermal tissue
« on: July 15, 2008, 06:24:16 PM »