Although I'm sure it works well for you, I don't like this control. I think it is comparing apples to oranges. I think many investigators will misinterpret their data using this type of "negative" control. What if the autofluorescence of your monos goes up after activation? What if Fc binding goes up after activation? in these cases your unstimulated monocyte negative control would be "negative" and your false positive would be read as a "positive" for IL-10. You really need to use the same cells that your cytokine specific mAb is being used on. One of the best controls is to block staining with unlabelled anticytokine mAb. Every investigator should use this type of control several times in pilot experiments to clearly demonstrate the specificity of their staining. Isotype controls are not sufficient. In a sizable fraction of the intracellular cytokine staining manuscripts I review, the signal the authors are referring to consist of noise (autofluorescence, Fc binding or non-specific binding). I think investigators should be increasing the rigor of their controls not making them more lax. > ---------- > From: Hodge, Greg (HAEM) > Sent: Monday, October 30, 2000 4:19 PM > To: Cytometry Mailing List > Subject: RE: in case of isotype control - substitute? > > > > > > ---------- > > From: Maciej Simm > > Sent: Tuesday, 31 October 2000 1:17 > > To: Cytometry Mailing List > > Subject: in case of isotype control - substitute? > > > > Maciej, > > > > I dont bother with isotype controls for IL-10 production by > > monocytes, I find unstimulated monocytes do not make any IL-10 and so > use > > this as my "negative" control. > > > > Greg Hodge PhD > > hodgeg@mail.wch.sa.gov.au > > > -------------------------------------------------------------------------- > > ---------------------------------------------- > > > > On 31 October, Maciej wrote- > > > > Howdy Everyone, > > > > I am running IL-10 in human monocytes using a PE conjugated Rat anti > > Human IgG1 antibody. There is a shortage of intracellular isotype > > control for that. What can I use instead? someone recommended using > > anti rat IgG2 etc.. but I've read on here earlier the 2's are picked > > up by monocytes.. > > > > so how about a mouse IgG1? > > > > Maciej > > > > __________________________________________________ > > Do You Yahoo!? > > Yahoo! Messenger - Talk while you surf! It's FREE. > > http://im.yahoo.com/ > > >
This archive was generated by hypermail 2b29 : Wed Apr 03 2002 - 11:56:12 EST