microparticles caracterization

llejeune@ldi.jgh.mcgill.ca llejeune at ldi.jgh.mcgill.ca
Thu Nov 17 12:30:30 EST 2005


Dear flowers

we try to caracterize and quantify microparticles from Endothelial cells.
we followed the protocols mentioned in the litterature (i.e. combes V and 
al, Journal of Clinical investigation, 1999 - Shet AS and al, Blood, 2003 
etc ...) 
Once in a while we are able to caracterize them according to what has 
already been described but we fail to have reproducible results.
All our media, FACS buffers, in general any liquid reagent has been 
filtered 0.22 micron

Our main problems are the following : 
1- the aspect of our microparticles (MPs) in FSC/SSC as well as the 
apparent concentration change with time (fresh vs stored samples) and 
sample temperature (on ice vs room temperature) , is there any specific 
way to store MPs or do we have to work with fresh sample ? What is the 
impact of the temperature ?

2-We have problems having a positiv staining for CD31, sometimes it works 
and sometimes not, it seems to be dependent of MPs concentration so we 
tried several concentrations of Ab for each experiment which seems to help 
sometimes, but not always.

3-For quantification, we run samples containing calibrated beads (250 000 
per sample), when we run beads only, (of course same concentration) we 
have a certain rate for beads, let's say 2000 per second, and when we run 
beads + MPs, we drop to 500 beads per second at the same speed	and this 
is very reproducible !!!... How can we explain that ? 

Thank you all for your help
 Laurence LEJEUNE
Flow cytometry facility - Rm 136
Lady Davis Institute
Jewish General Hospital
3755 Côte Sainte Catherine
Montreal H3T 1E2
Quebec Canada

(514) 340 8222	ext 5172
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