Phalloidin-Alexa Fluor 488 staining problem (huge background) - (Jul/10/2012 )

I am trying to set up a simple flow cytometry-based actin polymerization assay that relies on staining with Phalloidin, coupled in our case to Alexa fluor 488.

The assay doesn't work on any cell type, even those routinely used in chemotaxis assays in our lab. There is a huge staining signal for all cell samples, treated and control. I am thinking that Phalloidin staining is more difficult and touchy than the protocol we're using lets on. Does anyone have any insight?

The protocol was, briefly:

Stimulate chemokine receptor-bearing cells with chemokines for various times (by fixing aliquots of the stimulated cells at intervals)

Spin down, resuspend in triton-100X buffer, incubate for 20 minutes to permeabilize

Spin down, resuspend in 110 nM Phalloidin-AF488, incubate (dark of course) 30 min to stain

Wash 3X in PBS with 1% BSA (all buffers had 1% BSA throughout)

the protocol sounds fine to me. Alexa 488 is also a very good label compared to other products that work in the same wavelength. Your problem must be either setting up the machine, or your buffers.

Do you mix your sample with sheath buffer? I have to tell you that if I didn't mix my samples with this FACS buffer I wouldn't get reliable data.