With regards to the protocol by 10X Genomics: https://assets.ctfassets.net/an68im79xiti/4cs4w2bjmwUQsiNzwufhkv/e1c7b74aa9f10111090030246efe733f/CG000149_Demonstrated_Protocol_CellSurface_Protein_Labeling_Rev_D.pdf
I would like to clarify what's the purpose of having 10% FBS in the cell wash buffer for low viability samples? Does the FBS help the cells to remain viable longer? Or does the FBS act upon the dead cells?
Edit: I typically use 0.5% to 2% BSA in my wash buffers. So I'm mainly curious as to why it's recommended to use FBS instead in low viability samples.
Hello Ray Wang
Ideally for this protocol, the input cell suspension should contain more than 90% viable cells. Non-viable and dying cells may generally contain more fragmented RNA that may not be efficiently captured by 10x Genomics Single Cell Solutions. The presence of a high fraction of non-viable cells in the input suspension may therefore decrease efficiency.
Therefore, to maintain cell viability, particularly when samples contain less than 70% viability, or those samples that have low cell input, it is essential to have 10% FBS in the cell wash buffer. FBS does not act on dead cells.
Best.
In my view, since it is single cell suspension, you want cells should not stick together in clumps. FBS helps them not sticking to each other. On the contrary, if You use no FBS, cells will clump together and your purpose of single cells will not be accomplished.
Hi Ray Wang
The advantage of using FBS is its lower antibody content and higher growth factor levels which will benefit the cells. Moreover, BSA is a major component of FBS. So, it is better to use FBS in cell wash buffer, particularly when samples contain less than 70% viability or those samples that have low cell input.
I hope this explanation will help.
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