I tried to measure cell lysates and cell culture supernatants in differentiated MC3T3 using mouse RANKL ELISA kit (#EMTNFSF11, Thermofisher). While the cell lysates gave me a consistent reading (300-700pg/ml RANKL depending on stage of differentiation), I can't detect any RANKL in the cell culture supernatant.
I have tested the supernatant of complete medium (with 10% FBS), also the FBS free supernatant (switched to no FBS medium 24 hrs before measurement). Can anyone suggest the what might be the possible cause?
You may also try to concentrate your conditioned medium using centrifugal filter. We use Amicon Ultra filters from Merck.
Hope it will help.
Good luck!
Thanks you for all your helpful suggestions, will definitely tried to extend my culture time and concentrate my supernatants.
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