RAW264.7 Differentiation & TRAP Expression
Posted 05 November 2012 - 08:14 AM
Has anyone else had a problem with RAW264.7 differentiation or TRAP expression? What could be causing this? Or is there a way we can fix this?
Thank you for any suggestions you can give!
Posted 06 November 2012 - 12:17 AM
Have you tried fresh cells/ cells from a new source?
Have you changed anything in the culture protocol?
Have you tested for mycoplasma?
Does the morphology of the cells change as usual when differentiating?
Posted 06 November 2012 - 08:23 AM
We have tried different lots of RAW264.7 from ATCC, and all cells were negative for mycoplasma. The protocols have not changed (although we have now been trying to play with them to troubleshoot this issue). We often get the same morphology of the cells when differentiating, although in some assays we have seen very little differentiation while others are differentiating far more than we saw before. We have been testing new lots of FBS to see if there has perhaps been degradation of something that was aiding in osteoclastogenesis, but currently the new serum has had little effect.
Do you know of anything that might inhibit TRAP itself? Or overall differentiation? We have been told that LPS can negatively effect RAW cell differentiation, but when we followed up on the reagents we were adding they had very very low endotoxin levels. Is there something else we might be missing?
Posted 06 November 2012 - 11:41 AM
I would look a little more closely at the serum, there can be quite big batch differences, and a freeze/thaw cycle can have a big affect on the end result.
Other than that, I'm out if ideas sorry.