Particle sorting request

O'Connor, Eric eric.oconnor at csc.mrc.ac.uk
Mon Jun 30 07:51:59 EDT 2008


Hi Tim,

 

I looked into it and we used to sort a variety of influenza virus that
was described in literature as among the smallest bacteria, 200-300
nanometers. And yes we triggered off Log side scatter.	 We used a
VantageSE DiVa beta version to do it and used Biosure (Grass Valley, CA)
as a supplier of Sheath Fluid, it comes as a concentrated stock and has
negligible granularity.  What instrument(s) do you have?  Though use of
an ultracentrifuge is an option, I feel that the small volumes produced
per spin aren't realistic for generation of large amounts of sheath
fluid.	I'm not sure which model of ultra-fuge you've got, but ours
holds 8 or 12 (can't remember) 50mL Falcons.  We spin 2 hours for
DNA...so I'm not sure how long you would need to spin to get rid of
'space dust'.  

 

I assumed that you would be using more than one Biotin molecule per
particle to detect your Alexa Dye. If there is not biotin per particle,
you could also use an amplification system where you use naked
streptavidin to bind to the biotin molecules, and then come back with
anti-avidin, and then a biotin conjugated fluorochrome.  I believe this
is how I did it but it seems a long time ago.  You can repeat this step
again although steric hindrance may become a problem.  I this kind of
system (although I'm sure I wasn't the first) for detecting pictogram
amounts of cytokines in ELISAs.   

 

Here is the reference:  

 

O'Connor, E., E.D. Roberts, and J.D. Davies. 1999.  Amplification of
cytokine specific ELISAs increases the sensitivity of detection to 5-20
picograms per milliliter.  Journal of Immunological Methods.  229:
155-160.

 

Though not exactly the same concept, it could be useful for you.  Good
luck!  Let me know if I can offer any more help.

 

'The simplest approach is the best one'

 

 

Eric O'Connor

Head of Flow Cytometry

MRC Clinical Sciences Centre

Imperial College Faculty of Medicine

Hammersmith Hospital Campus

DuCane Road, London  W12 0NN

 

Tel: +44(0) 208 383 8330

Mobile: +44(0) 781 575 7730

________________________________

From: Mario Roederer [mailto:roederer at drmr.com] 
Sent: 27 June 2008 01:23
To: cyto-inbox
Subject: Re: Particle sorting request

 

That's a trick question.  If you want to know the proportion of beads
with any amount  of SA attached, the answer is no -- a single SA
conjugated to Ax568 can't be detected by standard FACS.  If you are
trying to distinguish beads that have a good number of SA attached (say,
100-300) from blank beads, the answer will be "maybe".	That depends on
whether you can reliably detect 200 nm particles by scatter -- the
answer is most probably not, but you can try (use log-side scatter as a
trigger).  If the beads are sufficiently coated with SA, then you can
trigger on the Ax568 fluorescence, and enumerate the positive beads.  If
all beads are intrinsically fluorescent or labeled (say with Ax488) then
you could trigger on Ax488 and count the ones that also have Ax568 vs.
those that have no Ax568 fluorescence.

 

Years ago, we use to analyze subcellular organelles by FACS, using
log-scatter as a threshold (you really need to use sheath fluid that has
been ultracentrifuged), or triggered on nile-red fluorescence (NR goes
into membranes) and quantify the other fluorescences... the organelles
were clearly in the sub-micron range, but it's hard to know how small
they got.

 

Sorting is trivial if you can detect them.

 

mr

 

On Jun 26, 2008, at 10:17 AM, Timothy Overton wrote:





Dear All,

 

I just has a request from someone working in my department which I think
is theoretically possible, but I wondered if anyone has tried anything
like it. They have nanoparticles coated in biotin, to which are attached
Streptavidin conjugated to Alexa 568. The size of the particles is
around 200 nm across. They asked whether flow cytometry could be used to
determine the proportion of particles with Strep-Alexa attached, and
then if they could be sorted. Has anyone done anything like this?

 

Thanks,

 

Tim

 

 

*****************************************

Dr Tim Overton

Lecturer in Biochemical Engineering

School of Chemical Engineering

The University of Birmingham

Birmingham B15 2TT

t: +44 (0) 121 414 5306

e: t.w.overton at bham.ac.uk

 

 

 

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