To study the toxicity of a contaminated soil with petroleum and heavy metals using human cell lines as model organism, we need to perform a pollutant extraction step before carrying out the experiments. After searching in the literature, we have prepared a protocol consisting in first step of mechanical extraction with a mixture of dichloromethane: methanol (2:1) and, after centrifugate the samples to separate the soil from the solvent, the latter is recovered and a second step of evaporation using a rotary evaporator is performed. Finally, we need to resuspend the obtained crude extract in DMSO. We are facing some problems in this last step, since we can not resuspend this extract completely in the DMSO (it sticks to the sides of the tubes).
Any idea to completely resuspend the contaminant crude extract in DMSO, or any other methodology to obtain the pollutants for this kind of toxicological assays?
Try sonicating the tubes.
see
Article Analysis of 51 persistent organic pollutants in soil by mean...
In general, we resuspend in acetone or hexane, but this is for GC analysis. Maybe you can try to add the small % of acetone (e.g. 0.05%), and use the same mixture for your control in the experiment. However, I don't know the effect on cells.
Article Analysis of 51 persistent organic pollutants in soil by mean...
Dear Carlos,
what I can suggest is to add a small amount of extract to the tubes in order to limit the "height" of the extracts in the tube. In this way, you will limit the spread of the sample onto the tube walls. You have to repeat the operation several times using only the sample or the solvent you used to extract the sample.
Our protocol includes the extraction, the evaporation with rotavapor to reduce the extract to 10 mL, and then the evaporation to dryness using nitrogen and needles (like this one https://it.vwr.com/store/product/582631/concentratore-di-campioni-sbhconc-1). Finally, we resuspended with DMSO using sonicators to try to dissolve all the materials.
The amount you will recover depends on the organic fractions (mostly total organic carbon) of the samples. In my works, sometimes I "created" very concentrated stocking solutions, in other case I have re-designed the experiment considering the higher equivalent concentration achieved by the experimental condition. Moreover, you can consider also the resistance of your cell lines to DMSO in concentration higher to 1% v/v. In this way you can dissolve the materials more easily (with higher volume) improving the solubility and recovering also the materials on tube wall. Some cells tolerate 2-3% of DMSO v/v.
You can also work on purified extracts if you want to focus on specific hydrocarbons family. In this way, you will destroy most of the undesired matrix and you will able to concentrate your portion with the compounds of interest.
I hope these suggestions will help you. Let me know.
Diego
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