rat bone marrow mast cell maturation - Anyone have any experience or advice? (Feb/06/2008 )
I'm trying to culture rat BMMC's (from Brown Norway rats) using similar culture conditions to those we've used successfully with mouse BMMC's, but with no luck. My current media is pretty standard - RPMI, b-ME, 10% FBS, P/S/glut, sodium pyruvate. rrIl-3 added fresh, tested with and without rSCF. also tested ConA stimulated spleen conditioned media, as a source of IL-3, but my cells seemed to stick to the plate and die faster than previous cultures. I only change out half the media with each feeding, as with mouse BMMC's or the viability decreases. Very few problems with viability - routinely >90% even after 35 days in culture. My main problem is that all of my cells keep adhering to the plastic - i've looked at the adherent cells by flow and spun some out onto slides and they appear to be monocytes / macs, which i don't want. All the suspension cells dwindle away quite quickly.
Any advice would be most greatly appreciated.
I don't know much about mast cells in particular, but IMHO IMDM with 5% serum seems to be overall better for growth of hematopoietic cells than RPMI or DMEM. The added benefit is that you only need 5% serum so less serum growth factors that may be impinging on your cell lineage committment.