Dilution of Inhibitor Using DMSO

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musicmaniac0428
musicmaniac0428's picture
Dilution of Inhibitor Using DMSO

 
So I'm looking to dilute my inhibitor from a stock solution of 5mM into the following concentrations for a WST-1 Assay run in triplicate 0.2μM, 0.39μM, 0.78μM, 1.56μM, 3.13μM and 6.25μM. I'll be adding 1μL of each inhibitor with 99μL of media to the wells and therefore would need to make up dilutions of 20μM, 39μM, 78μM, 156μM, 313μM and 625μM. I’ll be diluting the inhibitor with DMSO, however I need to maintain DMSO concentration at 1% in each of the wells. I will be treating the cells every 24 hours for 3 days and therefore need at least 9μL of each dilution so I want to make 20μL of each. I'm not entirely sure how to do this so I was hoping someone could walk me through the process/calculations to perform a serial dilution on this inhibitor while maintaining DMSO at 1% in each well. 

Sami Tuomivaara
Sami Tuomivaara's picture
musicmaniac0428,Here's one

musicmaniac0428,Here's one way to do this:Dilute 6 μL of the 5 mM (= 5000 μM) stock solution (inhibitor in DMSO) with 42 μL (7-fold volume) of DMSO. The inhibitor concentration of this first working solution is then one eight of 5000 μM, or 625 μM (in total volume of 48 μL).Then, prepare a new tube from this 625 μM solution by taking 25 μL of it and diluting to half with 25 μL DMSO. Now you have a second solution with total volume of 50 μL with 313 μM cocentration, and you still have more than 20 μL left of the first working solution.Then, take 25 μL of the 313 μM solution and dilute with equal voume of DMSO.And, repeat making one-half dilutions by mxing equal volumes of the previous inhibitor dilution and DMSO until you have series of tubes of inhibitors with concentrations 625, 313, ..., 20 μM.After these steps, only thing you have to do is to take small aliquot from the tubes and add 99-fold volume of the medium. Final concentration of DMSO in all the wells will be 1%.Cheers,