First Q: Most protocols allow for 1-5% FBS in FACS buffer solution, however, I use 10% FBS and I never had problems with the sort. what is the reason behind the reduced FBS?
Second Q: Is it okay to use alpha-MEM as a sort buffer? I have not encountered any protocols using alpha-MEM, most protocols use a standard sort buffer. My cells self-express GFP, Does have sth to do with the type/color of the stain used?
I also recommends Carter J Barger. Go reduce the chance of clumping less concentration of FBS is used.
Thank you everyone for your answers.
I always use 10% FBS with no problems however, I will try and use 1-3% and see how would this affect the results.
This is correct, chelating divalent cathions during sorting is a prerequisite. Besides why using a higher concentartion of FBS when lower amount allow a good cell viability ?
For most applications, 2% heat inactivated FBS is fine. Adding 2 mM EDTA can reduce cell clumping and reduce cell death, particularly with difficult samples/primary cells. Higher FBS % may affect the phenotype of your cells (obviously this depends on your cell type and staining), especially if your protocol involves long incubation times and higher temperatures (RT / 37°C).
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