I am simply adding Human Serum Albumin ( 5%), Mannitol 5% and Polysorbate 0.1% to RAW-Blue cell line and assessing cell viability (WST-8). I see cell death/decreased cell viability. Just adding albumin or Mannitol (a sugar), should not kill cells right? Why do you think this is happening? Cells are plated at 100,000 cells / well in 96 well plates.
100,000 cells per well in 96 plate is a very dense. I am seeding big cells in density 3000-5000 cells/well or small cells 10 000/well. Maybe this is the reason? Secondly adding excessive non toxic content can change the osmomolarity of the growth medium, what may have deletorious effect on the cell line.
If decrease in cell number is after incubation of cells for a day or two, it could be due to cell crowding as noted by Agnieszka. If it is within few minutes, it could be due to alterations in osmolarity differences. An examination under the microscope could reveal the later.
I agree with Agnieszka that the cell density is probably too high and/or you induce a rapid change in osmolarity. Two questions :
Why did you add mannitol in your medium ? you may compare adding either serum alone (toxin-free) or mannitol alone.
What is the time course of your death process, is it immediate or rapid (< 2-3h) or slow and progressive ? such time schedule can give you an idea concerning the putaive mechanism of cell ceath
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