Hello Everyone!!!
I am trying to differentiate RAW264.7 cells (1million cells/1ml/ well of 12well plate) using 10ng/ml or 50ng/ml of RANKL concentration for 5 to 7 days. Later on the TRAP staining with with 387A-1KT kit from SIGMA. But unfortunately I am unable to see TRAP+ cells under light microscope. So now I wonder is it a problem of differentiation protocol or staining protocol?
Can anyone share their established/in use protocol for both of these?
Thank you in advance!
HEy!
You are using way to much cells. They don't differentiate when they are to dense. I use 12.500-25.000 cells/cm². RANKL 50ng/mL is fine, but make sure it's a good source. In my experience R&D system RANKL is the best, of the cheaper products you often have to use much more. Also start differentiation directly with the seeding, no waiting or they get to dense, too.
Best,
Juliane
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