I'm currently differentiating CD34+ cells into macrophages using medium that includes m-csf and flt3. The differentiation works well and takes 14 days to get ~65-75% confluent attached cells in a T75 that look happy but once I lift, the cells lose significant viability, or completely die and the unhappy alive ones do not reattach to wells or glass slides.
I've tried various detachment methods including 5 minutes in cold 10mM EDTA in PBS, accutase, vigorous pipetting or cell scraping.
Has anyone had good success lifting and reseeding specifically primary macrophages ? Are there any imaging glass wells that I could differentiate the cells directly in that would keep them alive for 2 weeks ?
Dear Jeandele Elliot ,
for lifting primary macrophages I recommend Costar ultra low attachment plates, but of cause you can also differentiate your cells on glas bottom dishes (170 µm) like ibidi µ-dishes or WPI's FD-35.
best wishes Soenke
Hi,
I had similar issue previously, and PBS+EDTA worked good in my hands. I recommend spinning down the cells after lifting at 1200 rpm for 5 mins to get rid of dead cells. Also, re-plate the cells in the well plate/glass slides (glass bottom) that are coated with some adherent molecule. Fibronectin can work for you for coating the surface, that can help in adherence of the cells.
Good luck.
- Badges
- Science method