Sam,
I have to agree with Robert Z. All my instruments are B-D's; although I
was originally trained on an Coulter
Epics. I give you that background, because the alignment beads I use are
Coulter's Flow-Check Fluorospheres. I set-
up a start-up panel with the following displays: X=SSC vs Y=FSC[I told you I
was trained on Coulters], X=Fl-1 vs Y=
Fl-2, X=Fl-2 vs Y=Fl-3, and five histograms FSC, SSC, Fl-1, Fl-2, & Fl-3;
each with very small with M1 in each hist-
ogram display. My voltages remain constant and are saved as well as the
stats for each display. In this way I can
check if it is me or the instrument or both are off. The beads have been
very consistent and it is easy to check lot
against lot.
One of the problems I have with Chick Cells besides for not being
consistent is at times they have a tendency to
clog, and I'm not sure if they can be sterilized or not. Here I can be
corrected I've never tried. Hope this helps
The other
Howard
Howard S. Mostowski Core Mgr. Flow Cytometry
FDA/CBER/DCGT
Mostowski@CBER.FDA.Gov
[301] 827-0704
-----Original Message-----
From: zucker.robert [mailto:zucker.robert@EPAMAIL.EPA.GOV]
Sent: Tuesday, May 01, 2001 10:26 AM
To: cyto-inbox
Subject: Re: Laser Focus on Stream in Air
HI Sam
I have always questioned using a biological particle like a CRBC that is
NOT spherical to calibrate and align a flow cytometer. I understand that
this is the BD approach as they had this famous San Jose chicken that gave
them great blood cells. However, particles ( beads ) from various suppliers
are more reproducible and the data on machine perfoamce can be compared
between laboratories. However the criticism of these particles is that the
fluorescence of the alignment beads is usually high than cells and the
refractive index is different from cells. However, in our hands we have
found this the most reliable particle for alignment purposes. At least you
can get a CV value that can be compared between machines and with one
machine over time. The data from the CRBC is very subjective due to
biological variations, orientation factors and fluidic factors. I just
don't understand why more laboratories don't use alignment beads on a
routine basis to access machine performance. Perhaps someone on the Flow
list can enlighten me.
Bob
Robert M. Zucker, PhD
U.S. Environmental Protection Agency
MD 72
National Health and Environmental Effects Research Laboratory
Research Triangle Park, North Carolina, 27711
Tel: 919-541-1585; fax 919-541-4017
e-mail: zucker.robert@epa.gov
"Witherspoon,
Sam" To: Cytometry Mailing List
<sw11527@glaxowel
<cytometry@flowcyt.cyto.purdue.edu>
lcome.com> cc:
Subject: Laser Focus on
Stream in Air
04/26/01 05:03 PM
Greetings All,
I am always happy to learn new things, (thanks Yuri) and so I am
asking for input on the following:
For years I have been adjusting the beam *focus* on our sorter
with
help from a pulse width signal, in this case, FSC width. The idea is that
the smallest width represents the smallest beam spot.
*I use glut.-fixed chicken red blood cells on a FACStar Plus. (100u
nozzle)
with an elliptical spot.
*488nM laser at 60mW.
*The CV's I get on PI-stained CTN are 3% at 256 channel resolution, which
I
believe for this type of instrument is quite good.
Someone has my 2nd edition Shapiro tome and the cytomut treatise is
therefore not available to me now.
Is my reasoning on target? Constructive comments please.
Cheers,
Sam
Sam Witherspoon
sw11527@gsk.com
Tel. 919-483-3078
GlaxoSmithKline R&D Page 919-857-7768
5 Moore Dr. Fax 919-483-0585
RTP, NC 27709
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