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RAW264.7 Differentiation & TRAP Expression - (Nov/05/2012 )

I have been culturing and differentiating RAW264.7 into osteoclasts and measuring their activity using the Sigma TRAP Kit. Our protocols worked perfectly fine for the first 2 years I was conducting these assays, but within the past few months we have lost TRAP expression. Rather than getting a nice red substrate, everything just appears yellow. We have already contacted Sigma and gone through troubleshooting the TRAP assay and have found that the kit and reagents are working properly. We have also tried different kits that also have not worked.

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!

A

-an.forsyth-

Could be that the compound you are using for differentiation has degraded?

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?

-bob1-

Thanks for the response!
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?

-an.forsyth-

Hmmm, sounds like you have tried most things that I can think of. LPS can certainly affect differentiation, but as you say, the reagents should have low levels of endotoxins.

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.

-bob1-

Thanks for the suggestions.

-an.forsyth-