Hi im working on my thesis about hepatic steatosis...
I usually (SOP) overload my cells with palmitic and oleic acid...but just recently when i ran it through flow cytometry it turns out that my cells apparently were 'not stained'. I use nile red at 100ng/mL (10mg/10mL DMSO, taken 5uL dilute to 50mL = 100ng/mL); i incubate my cells at 37 degrees for 10 minutes in the nile red solution; around 2mL of 100ng/mL in 3 million cells/mL. There is absolutely no sign of stained cells in the flow cytometry experiment...please help?
in this experiment my superior told me that neutralizing trypsinized cells with 10%FBS in PBS is okay. is this the problem?
theoretically speaking if i prepared the stain as deascribed there should be some indication in the flow cy experiment but there isnt...can anyone help me with this?