FACS control high background - more stained cells in control than in sample (Aug/31/2005 )

Hi!

I just started working with FACS.

We work with mouse cells. The protokoll is established in the lab, but there is/was always a problem with high background in the negative control. The cell count in the control is often even higher than in the sample.

Control and sample come from the same Aliquot of a fresh blood sample with Na- Citrat.

Staining control: IgG2b-PE (anti-rat), IgG2a-FITC (anti-rat)
Staining sample: Flk1-PE (anit-mouse), CD117-FITC (anti-mouse)

We expect low rates of double stained cells, but of course not lower than in the control.

Any ideas?

i usually put some normal mouse serum (2%) during staining of murine cells to block non-specific binding sites

Seb_

I agree, you probably need to block Fc receptors with mouse serum or mouse IgG.

I agree with the other two responses and would add that the control antibodies should be the same isotype from the same species. You may already be doing this i.e. your anti-rat antibodies were not produced in mice?? and the Flk is a IgG2b and CD117 a IgG2a??

Thanks for answering.

I just realized Im made a mistake. Our controls are of course PE-Rat IgG Isotype controls and not "anti-rat"

But to look for the isotype was a good idea. I think somehow oders must be switched.

The PE-Rat isotype control was IgG 2b but the PE anti Flk-1 was IgG2a
and for the FITC antibodys the other way round: control IgG2a but anti-CD117 IgG2b.

I ordered new controls. Hope this will help.