We are currently trying to create a hepatocyte clearance method that can be used for compounds which are slowly cleared. To do this we are plating the hepatocytes on collagen 48-well plates (viability prior to plating determined using trypan blue). We allow the cells to adhere for 6 hours, change the media, leave the plates overnight and then the next day carry on with metabolic competency studies over a 24 hour period. The hepatocytes we use are these and we follow the supplier instructions: https://www.thermofisher.com/order/catalog/product/WICP10?SID=srch-srp-WICP10#/WICP10?SID=srch-srp-WICP10

While we are carrying out the metabolic competency studies we would like to determine how viable the cells are over the 24 hour period. We initially thought to use CellTiter Glo however, it doesn't seem as though it is lysing all of the cells in the well. We use CellTiter Glo according the the suppliers instructions - take the plate out allow it to equilibrate for 30 minutes at room temperature, add the CellTiter Glo in a 1:1 ratio and shake the plate at 90RPM for 30 minutes before measuring luminescence.

Manual for CellTiter Glo: https://ch.promega.com/-/media/files/resources/protocols/technical-bulletins/0/celltiter-glo-luminescent-cell-viability-assay-protocol.pdf

We were wondering if we could maybe add an additional lysis buffer alongside CellTiter Glo? To see whether that would help in fully lysing the cells over the 30 minute period. I've also read some papers online which talk about measuring Caspase 3 activity or Lactate Dehydrogenase... Wondered if anyone could shed some light?

I am a new researcher so please excuse my lack of experience..

Thank you :)