Re: flow sorting magnetic beads

I never sized dynal beads back in the days that they were the only choice, 
but they seemed to tower over the lymphocytes we used them to select in a 
micrograph, and would block my facscan (early nineties) - the advantage of 
those huge beads was that selection would work even for low antigen 
densities. From invitrogen's web site they now range from 1 to 4.5 microns, 
and would still affect the scatter of most cells should they bind.  The 
colloidal beads available from Miltenyi and possibly elsewhere have no 
apparent effect on scatter or flow through the machine (as long as you don't 
form precipitin with your antibody of course), but have seemed poor for 
negative selection in that they can't always pull a cell along - depending 
on the magnetic field, and they tend to leave a shoulder of stained but not 
magneto-motile cells, depending on the antigen density and field strength.

The ability to put the cells through a cytometer post-selection using 
colloidal beads led to an interesting artefact in my lab recently - the user 
positively selected using an APC-labelled peptide-loaded pentamer to a 
particular TCR re-arrangement to enrich "responding" cells in peripheral 
blood.	He then used APC-Cy7 to further phenotype the enriched cells.  He 
claimed that the APC-Cy7 must be breaking down, or that the machine was 
misaligned when all of his enriched cells (but not his unselected cells) 
were stained with the APC-Cy7 mAb.

It took a few passes through his protocol to get him to realise that the 
spare APC-binding capacity of his "beads" would capture APC-Cy7 conjugates 
regardless of the antibody they were  bound to, and cause his bead-enriched 
cells to appear to be APC/APC-cy7 double labeled.

As Rachael says the beads aren't magnetic unless in a magnetic field 
(they're just encapsulated rust) and won't aggregate in magneatically unless 
an external field is applied, but they may well stick together based on the 
antibodies that they're bound to and other proteins in the mix,


Cheers

Ray





----- Original Message ----- 
From: "Rachael Walker" <rvw24@cam.ac.uk>
To: cyto-inbox
Sent: Tuesday, December 11, 2007 10:41 AM
Subject: Re: flow sorting magnetic beads


Hi

Most magnetic beads that are commercially available are paramagnetic,
therefore will only be magnetic when in a magnetic field. Therefore the
beads won't affect the flow cytometer with regards to magnetism.    As
for the detection of the beads on the analyser it depends on which beads
you use.  Dynal beads are 0.5um in size, and in my experience I have
seen them on the machine esp as the beads seem to aggregate.   Whereas
Miltenyi Beads are smaller only 50nm in size and therefore won't show up
on a flow cytometer.  With magnetic beads it is often possible to do
negative depletion therefore the cells you want won't have any beads
attached to them and won't be activated in any way - important if you
separating cells such as  monocytes.  Also Dynal have a kit to remove
the beads from the cells  after separation.

Hope this helps

Rachael



Julia Sandberg wrote:
> Hello,
>
> I noticed that the last thread on flow sorting magnetic beads was from 
> year 2000, so I was just wondering if anyone has got any news regarding 
> this, since then? Do the beads accumulate in the FACS, or damage it? And 
> does the magnetism of the beads interfere with the sorting procedure? We 
> are using a FACS VAntage.
> I would be greatful for all input!
>
> Best Regards,
>
>
>
> ________________________________________________
>
> *Julia Sandberg*
>
> M.Sc.Eng. Ph D student
> Department of Gene Technology
> Royal Institute of Technology (KTH)
> AlbaNova University Center
> 106 91 Stockholm, Sweden
>
> Visiting address: Roslagstullsbacken 21, B3
> Delivery address: Roslagsvägen 30B
>
>
> Phone (office):     +46 (0)8 5537 8343
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> ________________________________________________
>
>

-- 


Dr Rachael Walker
Flow Cytometry Core Facility Manager
Wellcome Trust Centre for Stem Cell Research
University of Cambridge
Tennis Court Road
Cambridge
CB2 1QR

01223 760227