Re: Sorting very small samples

I'm not sure I see the benefit of contaminating a sample with  
"carrier" cells.  Why not just run the "carrier cells" first to set up	
the instrument, then wash out the fluidics with medium, then run your  
small sample (appropriately diluted).  Then there is no issue of  
coincidence, no possibly contamination.  If you dilute the small  
number of cells to the same volume as you would have with the  
"carrier" sample, then you will lose exactly the same proportion of  
the valuable cells during the initial fluidic stabilization... and  
your sort will be very slow, but at exactly the same rate as with the  
sample with the "carrier" cells.

The only benefit I can see to having "carrier cells" is to adjust  
settings during the sort -- but that's not a good idea to begin with...

I too can't wait for microfluidic sorters.... but I'm not quite as  
optimistic about your 4-5 year time frame.  I too hope to be wrong on  
this!

mr

On Jul 9, 2008, at 7:07 PM, Geoffrey Osborne wrote:

> Hi Simon,
> One strategy for sorting very small numbers of cells, in the hundred	
> to
> tens of hundreds range is the use of a "carrier sample" containing  
> cells
> with characteristics similar to those of the cells of interest yet
> spectrally tagged as a separate population (normally with something  
> like
> DRAQ5 these days). This allows you to set the instrument up correctly
> and yet avoid selection of the carrier cells. Then the critical step	
> is
> to run at event rates which dictate that coincidence occurrences are
> very low. If you have a super stable setup, which for some cell
> preparations is pretty routine, then the solution is to sort faster  
> BUT
> only single drop charges.
> Another point worth mentioning is that double or triple labeling with
> multiple fluorochromes to the same epitope to shift the population of
> infrequent cell of the "diagonal" of autofluorescence in multiple
> dimensions will also help allow you to pick a small population out of
> the carrier cells.
> We have used these approach many times over the years to sort very
> small samples.
>
> As to the microfluidics, don't hold you breath. Lots of potential, but
> based on my experience with microfluidic devices we make here it is  
> 4 to
> 5 years until we see a practical microfluidic sorter which can work  
> day
> in day out....but I'll be happy to be proven wrong.
> Hope this helps
> Geoff
> --
> Geoffrey Osborne
> Director of Flow Cytometry,
> The Queensland Brain Institute /Australian Institute for  
> Bioengineering
> and Nanotechnology,
> The University of Queensland,
> St Lucia, QLD, 4072, Australia
> Ph (61) 07 33466397
> email g.osborne@uq.edu.au
> Please consider the environment before you print anything!
>
> -----Original Message-----
> From: SIMON MONARD [mailto:smonard@staffmail.ed.ac.uk]
> Sent: Wednesday, 9 July 2008 6:33 PM
> To: cyto-inbox
> Subject: Sorting very small samples
>
> Greetings all.
>
> We would be interested in an instrument that is able to sort cells
> from very small samples, from a few hundred to a few tens of	
> thousands	
> of cells. Speed is not an issue. Are any such instruments  
> commercially	
> available? Does anyone have one we can try? I have seen a few talks
> over the years about microfluidics and on chip sorting but have never
> seen an instrument Any suggestions?
>
> Simon Monard
> FACS Facility Manager
> Institute for Stem Cell Research
> University of Edinburgh
> Roger Land Building
> West Mains Road
> Edinburgh
> EH9 3JQ
>
> Tel. Lab 0131 6505876
> Tel Office 0131 6517265
>
> -- 
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>