FACS positive cells in negative controls help - (Jun/12/2009 )

Hello,

I am beginner in flow cytometry. I am using FACS to analyze and sort adherent human cancer cells expressing receptor on the surface.
my problem is that lately i always see very high background in my negative controls. sometimes almost so high as positives in my samples :/

my negative control is lack of primary antibody (instead blocking in 5% milk) and then i add secondary antibody only.

i thought this was a problem with autofluorescence, but flow facility tech told me that patterns on my historgrams don't look like that. she thinks it is unlikely that the problem is autofluorescence, rather something else.
but what?

is it possible that i have so much of non-specific binding of secondary antibody? (i do not thing so, because in the past in my lab they did not have that problem and we are using antibody from the same company (invitrogen) forever). if yes, what to do? just try different antibody? or maybe i do not wash antibody out enough? i have no idea, what is wrong, but it drives me nuts.

do you have any suggestions what i can try? please, help

Hello Ania,
At the beginning, we get a lots of problems but they will be solved if the protocol is right.

If U could post the FACS profile we could have some idea. About using milk for blocking, is that the standard protocol followed for the assay U r doing? U can have isotype control or no primary antibody as control and compare the results. If U search around, there might be papers mentioning the methodology for FCM for the cell type and antigen. Did U have 'cell only' as a control? How was it with that?

Good Luck.

Nabi