negative control - (Apr/05/2010 )

i want to check the surface expression of a receptor in clones of a stable cell line. I just got confused if i should add antibody to my negative control? my negative control is the cell that does not express the surface receptor. And my positive control is the stable cell line that express the surface receptor.

I just confused wether i should add antibody to the negative control or should it be no stain? thank you

-soymilk14-

I would use both controls, unstained cells (cells that express the surface receptor, but no antibody) and stained cells that don't express your receptor.

-leelee-

leelee on Apr 6 2010, 03:58 AM said:

I would use both controls, unstained cells (cells that express the surface receptor, but no antibody) and stained cells that don't express your receptor.

Me too. Unstained cells will allow you to set the fluorescence so they are negative and cells that don't express the receptor but have been stained with antibody will allow you to gauge how specific your antibody is as you can see how much has stuck to the cells that don't express the receptor.

-than4-

i will do
for both stable cell lines (expess the surface receptor or not)
1. unstain
2. isotype of the antibodies
3. the targeted antibodies

with this u will have
FCM control(unstain-detect autofluorences, isotype-specificity of the antibodies) and experimental control( +ve or negative)
this may provide enough evidence to justified the result

cheers and happy flowing

a useful link
http://hsc.usf.edu/corefacilities/flowcyto...ow+controls.htm
thanks to flowcytometry core lab ,UNIVERSITY OF SOUTH FLORIDA for the excellent and informative webpage

-tongck-