Treating Hep G2 with drugs
Posted 14 April 2009 - 09:18 PM
I am planing to treat Hep G2 with different kinds of drugs(EtOH, paracetamol, phenobarbitone, WY14643, cisplatin and doxorubicin).
My question is what is the proper way to prepare the drugs. I have read in some articles that a stock of drug can be prepared in DMSO which can be stored in -20C and then a serial dilution by using media can be used to prepare the final concentrations---in other articles they prepare the drugs directly by dissolving the drug in media.....so I got CONFUSED
Should the media used for preparation of drugs contain FBS??
Any protocole for preparing the drugs will be great
Any help....in details plzzzzzzzzzzzz
Posted 15 April 2009 - 12:52 AM
If you need to dissolve in DMSO you must reduce the level of DMSO down to 0.01 % of the total volume of the media to make sure that this is not causing toxicity to your cells (DMSO can be toxic to cells at a higher concentration than 0.01%)
Also add a control set of wells to your experiment containing the highest concentration of DMSO only (i.e. 0.01%, if this is your highest conc.) This allows you to observe the effects that this conc of DMSO is having on your cells without the additon of the selected drugs.
To reduce the level of DMSO down to 0.01% for non water soluble drugs:
Dissolve a pre-selected amount of drug in DMSO (you should normally find solubility range on the drugs MSDS)
1)Perform a 1:100 (10 ul with 990 ul media) dilution of your stock with your media to get a 1% DMSO solution (this can contain FCS if you are only looking at cell viabilty/death as an end point
If you are looking at another end point you may need to remove the FCS if you think the factors it contains may have an effect on the results.
However you could also perform another set control set of wells with no FCS to see if their is a difference in your results
2)Perform another 1:100 dilution of your 1% drug/DMSO solution with your media, again with or without FCS if you need it,
This will give you your 0.01% drug/DMSO stock,
The actual concentration of your chemical in the DMSO depends on on how concentrated it was at the start as it will now be a 1:10,000 dilution of this as you performed two 1:100 dilutions,
So if you started with a 100mg chemical in 1 ml DMSO (100mg/ml stock), your highest working conc is 100mg/10,000 = 0.01 mg/ml or 10ug/ml. This is your new stock.
3)You can then use this stock to make up all your other dilutions using just your media
For water soluble drugs perform dilution exactly the same way but remove the DMSO at start and just dissolve in your normal media
Edited by cotchy, 15 April 2009 - 12:58 AM.
Posted 15 April 2009 - 04:34 PM
Posted 15 April 2009 - 06:42 PM
Thank you again and again................