What can I do to make my monocytes stay attached to my culture plate? - (Jul/10/2014 )

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?!

Hahaha, I detect some frustrations! :)

There are a lot of commercially available plates that are coated to facilitate cell attachment; think poly-D-Lysine coated plates, CellBIND plates (Corning) etc. Other than that, I know that changing medium may cause cells to attach to the culture plates. In my PC12 cultures, if I switch to DMEM instead of RPMI, the cells attach to the plate. According to literature, this is because of the higher calcium concentration (no calcium means cell detachment, it seems, at least in PC12 cultures).

Have you looked through literature to see if anyone else has ever tried this?

liguerr on Thu Jul 10 21:28:04 2014 said:

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.

I'm facing the same issue too, except with other type of culture. Hm... how old are the monocytes? Before you used them, were they stored properly at the right temperature? Also, have you tried centrifuging down a tube of culture and then pipette that into a plate? 

Thanks, Susie Q. I am going to try your suggestions and have ordered the plates. Fingers crossed!  

Coffenlucia, the commercially-obtained monocytes, I'm not sure, but those which I processed were only a week old, slow frozen in a propanol container placed in -80C then transferred to liquid nitrogen two days later. I thawed them a few days after that.  I'm not exactly sure what you mean when you refer to centrifuging a tube of culture and pipetting that into a plate. Generally from the liquid nitrogen we do a quick thaw at 37C, add media slowly, spin them, remove media, re-suspend them for plating in RPMI without FBS or pen strep. Then, plate about 1 million cells per 4cm2.