I do not know how big your particles are, but I have used the Beckmann Coulter
XL to count elementary bodies of Chlamydia Trachomatis following antibody
staining. However, I had to threshold or trigger on the green fluorescence as
the light scatter of those particles is way below the detection sensitivity of
the standard analysers, even of the modified optics of our Elite. If I remember
right Terry Prospero in Heidelberg / Germany did some work on synaptic
transmitter vesicles in the mid 80's but I do not know what happened with that.
The other problem you have to consider is the amount of background noise that
you can catch. Key requirement is to have clean solutions. When triggering on
side scatter for example you can detect a high number of background noise
(electronic and debris) which can completely hide your events of interest.
This can be particularly misleading in the case of a non-cumulative display
that only shows the last 1000 events as with the Becton Dickinson software. You
should then set the display to density plots.
Regards
Gerhard
-----Original Message-----
From: Rafael Nunez [SMTP:rafaeln@vetvir.unizh.ch]
Sent: Tuesday, June 13, 2000 4:18 PM
To: Cytometry Mailing List
Subject: Re: FACS analyis of exosomes
Hi Derek, we have used FITC for labelling and measurement protein content
in very small cells, like leishmania and E. Coli. In order to do that
measurement, we acquire the cells with log FSC and Log SSC. We set up the
gate on FSC and SSC and do histograms for FL1. It is very simple and is a
routine assay for our parasitology partners. Our paper describing this
measurement will come in Cytometry-August. In regard to equipment, a well
calibrated FacsCalibur can do the job, I hope this information will help
your "ambitious postdoc". Please contact me if you need more detailed
protocols.
Regards Rafael
>Greetings,
>
>I have an ambitious postdoc who would like to analyze the protein content
>of exosomes by FACS. He tells me that exosomes range in size from 50 - 100
>nM.
>
>Has anyone ever done this? or know if it is possible to do FACS analysis on
>"cells" this small?
>
>We have access to a FacScan, a FacScalibur, a FacsVantage and a MoFlo - any
>thoughts on which machine might be best for this analysis?
>
>Thanks for the input,
>
>Derek Sant'Angelo
>
>Derek Sant'Angelo, Ph.D.
>Assistant Member
>Immunology Program
>Memorial Sloan-Kettering Cancer Center
>1275 York Ave. Box 492
>New York, NY 10021
>Phone: (212) 639-6894
>Fax: (212) 794-4019
>Email: d-sant'angelo@ski.mskcc.org
\|/
(o o)
________________________________oOo__(_)__oOo_________________________________
___/\_ | Rafael Nunez mailto:rafaeln@vetvir.unizh.ch
/ o \/| | University Inst.for Virology http://www.vetvir.unizh.ch/
/ _| | Winterthurerstr. 266a Telephone: (+41) 1 6358710
/_/\__/-\/ | 8057 Zurich SWITZERLAND Faximile : (+41) 1 6358911
______________________________________________________________________________
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