Hello everybody,
I tried to isolated cancer cells from a tumor mass (xenograft).
I tried two different protocols where masses were initially digested with Collagenase + Hyaluronidase, in a second step with trypsin and then treated with DNAse and fitlered ... but I got only roughly 3x10^6 cells from a mass of 5x5x3mm3...
During the first washing step after the Coll/Hyalu digestion, I think I might have lost cells as after centrifugation the supernatant was still turbit/unclear... but I did not wanna centrifuge higher as usually when detaching cells to not stress them to much.
Does anyone have a tip ?
Hello Vanessa, from my experience with other cell cultures, the initial cultures are always with a low confluence. But from the details you gave, you may to take care with the trypsin, the incubation with trypsin should be not more than 10 minutes, because it damage the cells. I think this could be the crucial point in your protocol. I agree with your in the case of centrifugation.
Hi Vanessa, What type of tumor you are working with? I mean it might happen that the content of ECM or connective tissue in your tumor type is abundant than the cells. Try observing a H&E section of the xenograft which may give you an idea of cellular content
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