We got separation of the monocyte/macrophage population using ficoll, but the mouse hybridomas are still mixed with the human erythrocytes. We've tried magnetics beads for selecting the hybridoma membrane Ig for removal, but some hybridomas come along anyway. Has anyone used Percoll or other media for this?
Lee
You might be able to lyse erytrocytes specifically with red blood cell lysis buffer. I use this one for 1 minute on ice:
https://www.sigmaaldrich.com/catalog/product/sigma/r7757?lang=en®ion=GB
The usual procedure is lysing the erytrocytes .with a suitable lysis buffer A low level contamination should not be a problem for most applications. If you are going to grow hybridomas in vitro, erytrocytes wil die, so the culture will be totally clean. If the goal is purifying RNA to amplify hybridoma-specific genes, specific primers should guarantee the success after red blood cells' lysis to remove most of them.
There is something strange. Do you have human erytrocytes mixed with mouse hybridomas. Why are the species different?
Thanks guys !! I know, just lyse the erythrocytes. We have a co-culture propagating Babesia microti, a hemoparasite which grows in erythrocytes. So we are cleaning up the erythrocytes, not the hybridomas. It is time-intensive coating the hybridomas with magnetic beads for magnetic removal, but that's what we have available until some very clever scientist tells us how to do it the easy way.
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