I find that when I run an isotype control and then the labeled sample, the
negative population within the labeled sample is lower than the isotype
control. This results in my background marker to be higher than it should
be. Sometimes even cutting into my positive signal giving a lower percentage
positive population. Should I set my gate on the isotype control? or on the
negative population of my labeled sample?
I use the same titration of isotype control as in my labeled sample.
What would be a better background control? Using an isotype matched antibody
or using the unlabeled antibody of interest first, then the same antibody
bound to a fluorochrome in a second step?
ie; tube 1 - CD* pure or CD* Isotype control FITC
CD* FITC
tube 2 - CD* FITC CD* FITC
I understand that some isotypes (IgG1 vs IgG2a vs IgG2b) have a higher
background staining than others, can someone tell me how this goes for rat
and mouse isotype standards?
I appreciate any and all input regarding this issue.
Sharon Bader
Children's Research Center
Vancouver Canada
This archive was generated by hypermail 2b29 : Sat Mar 10 2001 - 19:31:06 EST