I am planning a cytotoxicity reduction assay and I have a doubt about the protocol.
First, I seed the cells for 24 h in a 96-well dish. Then, I pre-treat cells by adding my protective compound (200 uL per well) for 2 h. Later, I want to perform a co-treatment for 48 h by adding my cytotoxic compound (200 uL).
My question is:
Should I remove the protecting compound when the 2 h pre-treatment finish and later add 200 uL of the cytotoxic compound in the same well?
Or should I add less volume of my protective compound (100 uL) in the pretreatment during 2 h, and latter add 100 uL of the cytotoxic compound without removing the protecting compound?
Thank you for your time.
Hello David Vicente-Zurdo
You should first pre-treat the cells with the protecting compound for 2hrs. Remove the protecting compound and give one wash gently with PBS and then add 200uL of the cytotoxic compound in the same well.
Best.
Dear David Vicente-Zurdo
I would go with your 2nd option and add the first treatment at a lower volume (100uL). Since the pre-treatment is only for 2 hours I assume that the molecule either binds to something in the surface or enters the cells. In both cases washing with PBS will do very little to remove it. Also, washing cells itself is a treatment which may cause viability loss. Even though you wash all the samples including your non-treated cells, I would avoid extra stress whenever I can. I would only wash if I want to get rid of the vehicle (DMSO, lipofectamine etc.).
By the way, as reduction assays (such as MTT and WST8) cannot differentiate proliferation rate reduction from cell death, they cannot really provide evidence regarding cytotoxicity (only viability loss). I would prefer assays measuring membrane integrity (LDH, PI etc.) instead. Alternatively you can call your measurement "viability change".
For more info on this, I would recommend our article published on plos1: Article From viability to cell death: Claims with insufficient evide...
Cheers
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