Hi everyone,
I have been transfecting Ramos cells with my gRNA construct using an amaxa nucleofector and I proceed by sorting GFP+ cells (one cell per well) into 96 well plates. I also include a negative control of untransfected Ramos cells.
I have tried 2 times and the cells aren't growing neither the transfected or untransfected control cells.
I have been using the BD FACSaria for sort and it is set on the lowest/gentlest setting already.
And my media for the cells are 90% RPMI + 10% FBS
If anyone has any advice it would be greatly appreciated!
For single cell cultivation you should use "conditioned" media, for the protocol have a look at this publication .Article Assessing Somatic Hypermutation in Ramos B Cells after Overe...
You may include conditioned media for up to 40%, however, it is not always the case, I have been used either culture media alone or including conditioned media, the failing rates were similar; though, for what recommended by BD, it is more essential to increase serum percentage in your post-sort media, 50% of FBS should help to restore the health, than centrifuge and aspirate the cell into your culture media with 20% FBS is what gave me the most successful results.
Best.
Hello Nelly,
First as you control cells are not growing what sheath media is being used in your sorter? Ramos cells generally grow reasonable well after clonal sortings. Sorting from a single cell suspension resuspended in
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