Dear Friends,

I need some advice. Over the past year, I worked on osteoclastogenesis using RAW 264.7 cells. I never had any problems—everything worked well, and I consistently obtained a high number of osteoclasts.

However, for the past few months, since moving to a new lab, I haven't been able to generate proper osteoclasts, even though I’m following the exact same protocol. The cells look healthy and normal, but there is no OC.

Here’s what I’ve tested so far:

• 96-well plates (Corning/Costar, tissue-culture treated)
• Two different FBS sources: regular FBS and one specialized for differentiation (tested at 1%, 2%, 5%, 10% [standard], and 15%)
• High-glucose DMEM (Biowest) + Glutamine
• Two RANKL sources: R&D and PEPROTECH (the latter is the brand I used previously with success)
• Seeding densities of 4,000, 5,000, and 6,000 cells per well (passage 3–4, non-trypsinized cells)

Is there anything I might be overlooking?