J774.1 detachment for FACS - (Jul/01/2013 )

Hi,

I had read several post about this and have tried a few protocols to detach these sticky cells. My issue is I need to treat them with drugs, detach them and then run them through FACS. So membrane puncturing/disrupting protocols i.e. trypsin/scrapping are not appropriate.

Here's a list of my failed approaches:

1) Trypsin - left for max 10 mins - should not use for as puntures cell wall
2) Accutase - 5-30 mins at 37 oC - my cells pretty much disintergrated...
3) Citric saline (cold) - 10 mins room temperature only removed half the cells but was the best
4) On ice - 10-20 mins

I always tap the sides of the T75cm flask etc try to dislodge them but not luck. Like my father and his Sega mega drive they just dont want to let go.

Any advice will be gratefully received!

Failing advice for these cells can anyone suggest a macrophage cell line that are easy to remove from tissue culture flasks?

Ps. I do not have the money in my grant to buy a Biological Stirrer.

You could try TrypLE from Gibco.

Dear Aspergillus79,

You will know from my posts on J774.1 in the past that we use Techne stirrer bottles.......but you have no money.

My suggestion is that you culture your cells in non Tissue culture treated plastic. Try a few suppliers out and see which is the best....you may find that they very loosely attach and you can detach them just by shaking.

Good luck

Uncle Rhombus

Just wanted to edit my previous post, but somehow there was no edit button... ??

So here it is again (edited):
have you tried accutase at RT ? 5 to 30 (!!) mins at 37ºC sounds quite strong...