I'm trying to sort GFP positive DRG neurons and then re-plating them for imaging. The problem that I'm having now is that I'm unable to dissociate the culture effectively to run them into the Sorter. The cells seems to form a stronger matrix after 5 days in culture that could require a special mix of enzymes to dissociate them. Anyone has any idea how to do so ?
Thanks,
thanks, do you have a protocol for accutase that you can pass to me please. because on the website, it says 5 to 60 min incubation ?!
Hey Zainab,
I have used acutely dissociated lumbar DRGs for the patch clamp. I used collagenase/ trypsin-EDTA mix for proteolytic digestion and fire-polished glass pasture pipettes with the rubber bulb to mechanically dissociate them. This mechanical dissociation can be done gently, it worked quite well in my hand as I could get healthy and separated single cell for patch clamping. DO write me back if you need detailed protocol.
I hope it helps ! Best Nand
- Badges
- Science method