Hello Elisha Choong

1. After seeding cells in 96-well plate, do I need to remove the existing media from it before I add my treatment?

Yes, aspirate old media and add 100ul of treatment media to all wells and incubate as per your requirement.

2. Do I need to remove my treatment before adding MTT solution?

Working MTT solution can either be added directly to treatments or the treatment media may be aspirated and replaced with the working MTT solution.

Good Luck!

Dear Elisha,

I strongly advise you to remove treatment before adding MTT solution.

If your treatment contains any reducing agents, then these agents will react directly with MTT and will reduce it to the purple formazan crystals.

In other words, the MTT reaction will occur not only inside mitochondria of metabolically active cells, but also in the extracellular culture medium as well.

Hope I helped,

Chris

Christos Velesiotis Thank you so much for your advice! My current treatment only consists of DMEM supplemented with an ethanolic plant extract with very minimal volume of DMSO (for solubilizing purposes of the plant extract,