Re: Particle sorting request

Mario wrote:
> 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@bham.ac.uk <mailto:t.w.overton@bham.ac.uk>
A flow cytometer with a sensitive forward scatter channel, such as the 
Apogee or the Cytopeia/BD inFlux, should be able to detect and trigger 
on 200 nm particles, and many instruments should be able to use a side 
scatter signal as a trigger. Whether this will work with the 
nanoparticles in question depends on their size distribution (the 
narrower the better) and how much other stuff in the size range is in 
the sample and sheath.

As to fluorescence signals, people have sorted microvesicles in the size 
range mentioned, but they were probably looking for at least a few 
thousand fluorescent molecules/particle. I agree with Mario that a few 
hundred molecules might be detectable in a more-or-less conventional 
sorter under near-ideal conditions. You could probably get down to 
single cell fluorescence in a slow flow instrument, but the millisecond 
range dwell time would limit your analysis (and sorting) to a few dozen 
particles/second.

-Howard