I dissolved CCL4 in DMSO and added it in medium in a dose of 0,5 and 1%. However, oily spots (bubbles) are formed on the surface of cells.
Hi,
You can use a gama Glutamyltranspeptidase and bilirubin stimulated by hepatotoxic drugs or ethanol.
I do not understand your question. CCl4 is not protective. It is a zone 3 toxin. This means that it kills the hepatocytes that under low oxygen tension express the p450s (cyp) that are largely responsible for making insoluble molecules soluble. Can you be more specific on the foam (time course and appearance).
Aziz, I understood that you want mesure the hepatotoxicity, then I suggested use those enzymes. Would like now about the solution CCl4+DMSO?
If you want to measure cell death by necrosis, Joao is very correct. Measure the activity or amount of hepatocyte specific enzymes in solution by either initial rate measurements or westerns. I would avoid the bilirubin because even live hepatocytes will secrete so background will be high and dynamic range for observations will be small.
Thanks for all for your advices. May be I incorrectly formulated my question. My question is that I cannot achieve absolutely solubility of CCL4 in nutrient medium (how I can achieve absolutely solubility of CCL4 in nutrient medium?).
Well now your question makes sense to me. If you treat an animal with CCl4, mix it 1:1 with corn oil and ip inject. If you want to treat cells then make a 5 % solution of CCl4 with ethanol I suspect that the DMSO solvent will work well too but you need to go to lower concentration of CCl4 than you are using now. Keep the total addition of ethanol/DMSO below 2% of the culture. Be sure to have a EtOH/DMSO only control.
Your treatment with 0.5% is extremely high. The "foam" you see is likely the membranes being turned into "soap" via the free radical damage. Do a does response curve with whatever CCl4/EtOH stock you make to determine your LD50. 0.35 ul of CCl4 per ml of culture will lay waste to your hepatocytes in the order of 2 hours.
Good luck on your work.
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