difficulties in trypsonizing U937 derived macrophages - (Mar/14/2011 )
Hi, I'm currently trying to optimise my method to differentiate my U937 monocytes to macrophages. However I am finding difficulties when trypsonizing these cells. There seems to be a lot of cells remaining.
My procedure for differentiating is as follows
1) seed cells at 5X105/ ml,adding 10ng/ml PMA. Then adding 2mls of culture to each well in a 6 well plate.
2) Incubate for 48hours
3) Remove PMA and add fresh media
4) Incubate for a further 24hours
When trypsonising i remove all media then add 0.5ml to the well. I have left the trypsin-EDTA for as long as up to 7minutes, and have incubated the plate whilst the trypsin is on, but this seems to make no difference.
Any suggestions as to how long I should be leaving the trypsin on, and if I am doing this correctly?
You are doing fine. I suggest you before adding the Trpsin wash the cells with PBS as it removes the remaining serum from cell. As serum prevent the function of Trypsin too. And after adding the Trypsin incubate it in incubator.
Hope this work for you.