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
