I have been trying to culture monocytes forever (about six months). The first day, the monocytes . The next day, about 70% are floating. We always end up doing our IFN gene expressions ok, but the cell count is greatly reduced and we'd like more cells to be able to infect (it would be nice to have about 80% coverage of the plate surface). Here Is what I HAVE tried and has not worked. If someone can give me something else, I'd be open to it.
Trouble-shooting fails I have already tried with the same exact results:
1. Seeding monocytes on regular plates, RPMI media with no FBS, then waiting until they attach to add the media with FBS for a final 10% FBS Concentration.
2. Adding RPMI Media WITH 10%FBS
3. Leaving floaters for several days before removing them, hoping they would eventually decide to attach
4. Adding WAY more cells to the well than the protocol suggests
5. Adding WAY less cells to the culture plate well than the protocol suggests.
6. Using monocytes positively selected for CD14
7. Using monocytes negatively selected
8. Using collagen-coated plates
9. seeding them on charged glass chamber slides
10. Ordering commercially available monocytes
11. Isolating my own monocytes
12. singing to them, talking to them gently, pleading with them to please stay attached
Ok, what else is there?!