We are trying to separate tubular cells from mouse cortical kidney using a Percoll gradient without success. The isotonic solution we are using contains 2.5 mM CaCl2. Someone told me that Percoll gradients work better if the solution they are in is calcium free. I could not find any information about that and I was wondering what people think. Thanks!
Calcium is involved with cell:cell adhesions, which you do not want when performing density centrifugation. What is not working with the separation? What gradients are you using?
I suppoose, in presence of calcium cells can adhere to walls or other cells. Without calcium they simply cannot adhere to anything.
We are trying to separate proximal from distal tubular kidney cells without glomeruli. The papers I have seen say to use a 50% Percoll solution in Krebs-Henseleit buffer which contains calcium (Vinay et al 1981 Am. Physiol. Soc.). After centrifugation, we get glomeruli contamination everywhere, although it is said that the glomeruli should be on the top of the solution. We also don’t get a great separation of proximal and distal tubular cells. If calcium makes cells stick together, then that would explain it and I should figure out how to get an isotonic solution that is calcium free. Thanks a lot Even and Mike!
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