Most current commercial cytometers tail off at 500nm scatter
detection. trigger should be side scatter, preferential peak
signal.
Initial strategy is to use 500nm yellow/green beads from
polysciences. Dilute particles to reach approx. 10^6/ml (see
formula in their particle catalog). Look at green
fluorescence versus sisdescatter. Threshold on green
fluorescence with your normal lymphocyte settings.Once you
see the fluorescent signals lower threshold down to noise
level. Adjust scatter to have beads in the middle of the
side scatter scale. Change to side scatter triggering with a
setting at mid scale and reduce slowly. You should
eventually see non-fluorescent noise signals in the low
scatter area. Run filtered sample liquid as a control.
On the XL we routinely analyse 320 nm particles triggered on
peak side scatter, but it requires a clean sheath system.
Good luck
Gerhard
______________________________ Reply Separator _________________________________
Subject: Microparticle Analysis Using FACScan/Sort
Author: laurie_stojanovic@baxter.com at INTERNET
Date: 02/04/1999 20:15
Has anyone out there used a Becton Dickinson FACScan or FACSort flow
cytometer to analyze platelet- or WBC-derived microparticles (0.2-1.0 um in
size)? I am looking for assistance in the following arenas:
1. Distinguishing background (noise) events from "real" microparticle
events; looking for ways to increase the limit of sensitivity of
these instruments (Becton Dickinson Suggests limit is 0.5 to 1.0 um).
2. Setting used during acquisition; especially, threshold trigger
(FSC or FL) and values thereof
3. Gating strategies
4. Sample collection and staining
Please respond to laurie_stojanovic@baxter.com with any information you
have that could help me. I may also be reached at (847)270-4422.
Thanks!
Laurie Stojanovic
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