We are having a debate in the lab. Protein lysates are wanted from these frozen cells to run a cell signaling luminex assay.
I say if you are going to do this, you should plate and culture the cells for at least 24 hours to get them back to being happy, and then to isolate your proteins. This would give a better representation physiologically what the protein profile was in these cells.
Any input is much appreciated.
Hi Amanda,
I totally agree with you.
First, cells will be happier in a culture and need to remove the DMSO. Second, FBS contains too much proteins some that might affect the assay.
This surely depends on the cell line, but I think your 24 hour culturing period might be even too short to get them back to normal levels of gene expression and protein synthesis. Based on what we see with qPCR data from primary cells, I'd go for 72 hours (as long as you can recreate the conditions prior to freezing).
These are not lines. These were PBMCs drawn and frozen immediately after isolation.
For cell lines I would certainly agree with Stéphane's and Nora's suggestions, but in the case of snap-frozen primary cells I believe that culturing them first would change their protein expression profile much more dramatically than freezing does. Keep in mind that most proteins have a pretty long turn-over time, so the protein content of frozen cells should be fairly representative of the in vivo situation (disregarding protein degradation in the frozen samples for a second).
My suggestion would be to thaw the cells on ice, wash a couple of times with cold buffer to remove DMSO and serum proteins, and then lyse the cells immediately. If possible I would also include freshly drawn PBMCs as controls in the subsequent assays.
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