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Lysis of osteoblastic cell lines for dual luciferase assay

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#1 ocram



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Posted 31 October 2016 - 03:51 AM

I need to perform a dual luciferase assay on transfected MC3T3 cells, an osteoblastic cell line.  I have already performed this assay successfully on Cos7 cells, but I want to transfer this same assay to an osteoblastic cell line.  My problem lies in the cell lysis step, following 48h  transfections.  I have used various osteoblastic cell lines, but come up consistently with the same problem.  Since the cells form a collagenous matrix around them on the culture dish they are very difficult to lyse for the assay.  The assay  (Promega Dual Luciferase Assay) requires passive lysis, with the kit specific lysis buffer, but this buffer merely detatches the cells  and then clumps them together without lysing them.  I have tried treating the cells with collagenase (Dispase ) prior to lysis and then removing the collagenase.  The dispase works, but as soon as I add the lysis buffer the cells clump together again.  I am at a loss as to what to do,  since I need to eventually perform my experiment in a 48 or 96 well format, so I need to be sure that the lysis has worked!

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