Re: ADCC flow based assay

Hi Holly,

What you are suggesting is a feasible way of doing this and is the approach
we took a number of years ago. The labelling of the target cells with the
PKH-26 was the trickiest part and needed a bit of working up of the staining
conditions (time, washes etc). We wanted to use the orange one to keep a
green channel free for either an antibody or Annexin-FITC. One of the
advantages of TO-PRO-3 is that, in common with that family of cyanine dyes,
it can pick up early apoptotic cells as well as dead one if you incubate
slightly longer than you would just for live-dead discrimination. We found
that just using PKH and TP3 gave us the results we needed (and much more
quickly than other ADCC assays). It was also easy enough to add a method to
pick up apoptotic cells, annexin seems the best bet for that in this assay.
The assay itself is found here:
Lee-MacAry et al J Immunol Methods. 2001 252(1-2):83-92.
Wilkinson et al J Immunol Methods. 2001 258(1-2):183-91.

Good luck!
Derek



On 13/2/08 22:27, "Troche, Holly" <HTroche@dynavax.com> wrote:

> Has anyone tried a flow based ADCC assay and what were your experiences with
> developing this assay? I am attempting to do such an assay using PKH-26
> membrane dye to label the target cells, TOPRO-3 iodide to measure viability
> during the assay, and annexin-v FITC to measure the amount of apoptosis in
> the target cells.
>  
> Holly Troche
> Research Associate II
> Preclinical Research
> Dynavax Technologies Corp.
> 2929 Seventh St. Suite 100
> Berkeley, CA 94710
> htroche@dynavax.com| 510-665-0405


-- 
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Derek Davies, FACS Laboratory, London Research Institute,
Cancer Research UK, 44 Lincolns Inn Fields, London, UK.

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FAX: (44) 20 7269 3479
Mobile: 07790 604112
e_mail: derek.davies@cancer.org.uk
http://science.cancerresearchuk.org/sci/facs/

In tenebris lux    
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