Re: $$ Fees for flow? $$
From: PAUL@flowcyt.cyto.purdue.edu
Date: Wed Feb 14 1996 - 19:06:48 EST
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Since this $$ question comes up constantly, perhaps I should put up
the table Sue Demagio compiled a year ago on my web site? That way we
coudl keep a runnign commentary for people to check against? Any
problem with this Sue or others?

Paul
J.Paul Robinson, Purdue University Cytometry Labs
robinson@flowcyt.cyto.purdue.edu PH:317-494 6449 FAX:317-494 0517
web http://www.cyto.purdue.edu
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ThE data is already available on our website at Purdue the  numbers
for over 80 core labs in the country !

FIXING PRICES FOR ALL LABS THROUGOUT US...ON PURDUE MAIL LIST

Re: Canadian Fee Survey
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From: J. Paul Robinson <j...@flowcyt.cyto.purdue.edu>

Date: Sun Mar 18 2007 - 23:12:39 EDT
These data are already available on our website at Purdue
http://www.cyto.purdue.edu/flowcyt/labinfo/rates.htm  there are
numbers for over 80 core labs in the country  regards    paul
robinson      Kristin Chadwick wrote:  > Hello all,  >    > I run a
Flow Cytometry Core Facility at a Canadian academic institute.   > We
are currently evaluating our user fees for the upcoming fiscal year.

could you   > please indicate which of the following services you
provide, along with   > the associated hourly rates:  >    >   > 1)
Analyser  user operated  >   > 2)  Analyser  with Core operator
support  >   > 3)  Sorting  by

Core operator  >   > 4)  Sorting, or
use of sorter for analysis (ie UV, multi-colour not   >
possible on Analyser)  user operated  >   > 5)  Training  >   > 6)
Data analysis on offline workstation  >   > 7)  Do you distinguish
between academic/institutional or   > non-academic/corporate users?

is   > complete. Thank you in advance for your assistance.  >   >
Sincerely,  >    > Kristin  >   >   > *Kristin Chadwick*  > *Manager*

Robarts Research Institute  > P.O. Box 5015, 100 Perth Drive  >
London, ON  Canada  N6A 5K8  > Phone: (519) 663-5777 x34042  > Fax:
(519) 663-3789  > kchadw...@robarts.ca <mailto:kchadw...@robarts.ca>

Professor of Immunopharmacology & Biomedical Engineering  Director,
Purdue University Cytometry Laboratories  President, International
Society
for Analytical Cytology    Purdue University Cytometry Laboratories
Bindley Bioscience Center  1203 West State Street  Discovery Park,
Purdue University  West Lafayette, IN 47907-2057  Ph (765) 494 0757;
Fax (765) 494 0517  email: j...@flowcyt.cyto.purdue.edu  www.cyto.purdue.edu
Join ISAC - www.isac-net.org    Change lives today  - www.cytometryforlife.org
Received on Mon Mar 19 11:18:02 2007
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because I'm basically   too lazy to reply to each individual person
(sorry)) Enjoy yourselves,  Geoff  --  Geoffrey Osborne  Director of
Flow Cytometry,  The Queensland Brain Institute /Australian Institute
for Bioengineering   and Nanotechnology,  The University of
Queensland,  St Lucia, QLD, 4072,
Australia  Ph (61) 07 33469912  email g.osbo...@uq.edu.au
Received on Tue Mar 20 10:18:03 2007
Internet time scheduling system
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From: Geoffrey Osborne <g.osbo...@uq.edu.au>
Date: Tue Mar 20 2007 - 00:13:11 EDT
Hi All,  Due to the large amount of interest generated by my previous
post
regarding the FREE   multi language instrument time scheduling system
we use here, (and because I'm basically   too lazy to reply to each
individual person (sorry)), please find have a look at this  link
http://mrbs.sourceforge.net/index.html  which shows you where the
software comes from, and look here    http://mrbs.sourceforge.net/sshots.html
for some screen shots, which are about 7 years old...    Get your
local computer people to install it. I had my local IT guy modify it
to enable   booking "lock-out" at a configurable time prior to the
appointment time. This  modification is   available if I chase him up
to find out what he did.....    Enjoy yourselves,  Geoff  --  Geoffrey
Osborne  Director of Flow Cytometry,  The Queensland Brain Institute /
Australian Institute for Bioengineering   and Nanotechnology,  The
University of Queensland,  St Lucia, QLD, 4072,
Australia  Ph (61) 07 33469912  email g.osbo...@uq.edu.au
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DEVESTATIONS FROM LINEARTY

I have    > arrived to the conclusion this is due to faulty log-amp
behavior    > causing deviations from linearity. Certainly, this has
important    > consequences for our results on that machine!
Re: 2 machines, 2 compensation troubles.
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From: Howard Shapiro <h...@shapirolab.com>
Date: Tue Mar 27 2007 - 19:29:40 EDT
Uriel Trahtemberg wrote-    >  > As usual, when confronted with
trouble I turn to you for help. this    > concerns two machines with
similar problems.  >  > 1) The FACScalibur in our lab. I performed a
titration experiment    > for Sytox Green and when checking the
compensation needed for it to    > work with other stains, saw the
results shown in the pdf "SytoxG    > linearity". I have attached one
of the files (ApoPMN.NucAcTit.   > 1mar07.021) if you want to check it
out. As you can see, instead of    > being a nice distribution on the
X axis, it looks like a waving    > serpent! On close inspection, the
waviness can already be seen in    > the uncompensated FL1 vs. FL2
plot. After long thought, I have    > arrived to the conclusion this
is due to faulty log-amp behavior    > causing deviations from
linearity. Certainly, this has important    > consequences for our
results on that machine!
In order to confirm    > my findings I exported the FCS data to
excel, sorted the data    > according to the FL1 intensity and did a
FL1/FL2 plot. After   > accounting for statistical noise, the mean
ratio should stay   > constant - after all, that is the rationale of
compensation! As you    > can see from the "SytoxGreen.xls" plots,
that is hardly the case.  > I turn to you to ask for alternative
explanations and possible    > solutions. Is this kind of problem
repairable? how easily? We have    > a service contract with BD for
that machine, so would I be right to    > assume they should fix it?
What kind of tests would you suggest to    > confirm and/or refine my
findings?  > PS: SytoxGreen is NOT metachromatic AFAIK.    I'm feeling
lazy so I'll only comment on the first "problem", in    which the
reasons for replacing log amps in newer cytometer designs    become
apparent.    See p. 202 of Practical Flow Cytometry and the middle
panel of
Figure    4-57 (you can also go to www.analog.com and download
AD8307.pdf,    which is a data sheet on one of the newer log amp
modules). A log amp    is not a log table. The log amps used in most
flow cytometers are    made up of multiple modules and exhibit
linearity curves that are    wavy even in the range in which they
perform best. The typical   deviation from peak to trough over that
range is plus or minus 0.5    dB. Do the math and you will find that
is plus or minus 6 per cent,    if you're talking voltage. Plus or
minus 6 per cent is fine if you're    measuring immunofluorescence,
where other biologic and instrumental    factors typically contribute
much more to variance than does the    inherent, built-in, not-
correctable-by-replacing-parts inaccuracy of    the log amp. If you
measure nucleic acid stains, which are    stoichiometric, with a log
amp, you pretty much get a picture of the    log amp's nonlinearity
curve. The solution to the problem is to    measure
such stains using linear electronics. Most flow cytometer
manufacturers have now opted to build high dynamic range linear
electronics that typically allow accurate measurement over a four-
decade range; the resulting data are digitized to enough bits'
precision to allow conversion between linear and log scales to be
done digitally. It is, however, worth noting that Cytomation/Dako
and    Cytopeia cytometers do use log amps, converting the log data
with    high-resolution ADCs to allow digital linear-log conversion
for    compensation purposes, and the users of these cytometers are
not, in    general, bothered by the inaccuracy - unless, of course,
they try to    do something like measure DNA on a log scale.    -
Howard
Received on Wed Mar 28 13:58:00 2007
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in  Aria I can4t distinguish the peaks

Cell Cycle on FACSAria
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From: Katherina Garcia Garcia <kgar...@upo.es>
Date: Mon Mar 26 2007 - 10:00:57 EDT
Hello!    We have problems to see cell cycle on Aria. Sometimes we see
the 2 peaks but most of the  times we can4t.  We see the same sample
on Calibur and Coulter and the cell cycle is very good, but .    I am
using the right filter and I pass the sample at a very slow speed. I
use PI-A vs
PI-W to exclude doublets but I can4t do it because is imposible to
distinguish wich dots  are single or doblets cells.    Any ideas?
Thanks in advance.    Katherina Garcma Garcma  Ticnico de Microscopma
y Citometrma  kgar...@upo.es  Centro Andaluz de Biologma del
Desarrollo  Consejo Superior de Investigaciones Cientmficas
Universidad Pablo Olavide  Ctra. Utrera km1  41013 Sevilla  Espaqa
Received on Tue Mar 27 12:18:00 2007
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cloning but I had a hard time setting up the Aria
cell line sorting on Aria

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cloning but I had a hard time setting up the Aria
cell line sorting on Aria
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From: Julie Bertout <julie.bert...@ibl.fr>
Date: Wed Mar 28 2007 - 10:03:04 EDT
Hello,    We would like to sort cell line transfected with GFP in
order to do a   cloning but I had a hard time setting up the Aria...
No problem to sort
lymphocytes or eosinophils from blood or spleen in   high speed
sorting but the medium and low sorting required for cell line
sorting are difficult to set up.    Does anybody sort transfected cell
line ? what conditions do you use :   concentration of cells? events/s
recommended? amplitude usually used?   pressure (BD told me sometimes
you have to play a little bit with it...)?    Thanks for your
answers.    Julie Bertout  cytometry lab  Institut Pasteur de Lille  1
rue du professeur Calmette  59800 Lille  France
Received on Wed Mar 28 13:18:00 2007
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From: McCloskey, Thomas <thom...@nshs.EDU>
Date: Tue Mar 27 2007 - 14:01:06 EDT
A powerful way to analyze CFSE data is to calculate "precursor
frequency" or the % of the starting population which divided in
response  to the stimulus.  Some software can do this automaticaally
[such as  Flowjo].  If you have discrete peaks, you can also calculate
it  manually.  The idea is that in the one division peak, two cells
came  from one starting cell.  In the two division peak, four cells
came from  one original cell, and so on.  You end up with the number
of cells which  divided and by comparing to those cells which did not
divide [the first  peak or no division peak] , byou now have the
precursor frequeecncy for  that stimulus.     Good luck,  Tom
************************************************************************
*****  Thomas W. Mc Closkey, Ph. D.  Assistant Investigator, Center
for Immunology & Inflammation  The Feinstein Institute for Medical
Research, North
Shore University  Hospital  Assistant Professor of Pediatrics, New
York University School of  Medicine  Biomedical Research Center, 350
Community Drive  Manhasset, Long Island, New York 11030  ph:
516-562-4844 [office], 516-562-1084 [lab]
************************************************************************
*****        >>>-----Original Message-----  >>>From: Erietta Stelekati
[mailto:e...@fz-borstel.de]   >>>Sent: Monday, March 26, 2007 6:58 AM

all,  >>>I want to compare 3 pictures of CFSE proliferation. I can
see   >>>differences in the percentage of cells in each pick. But
how   >>>do I put   >>>all the data together and decide if it is a
statistically   >>>significant   >>>result? I can make a graph with
the percentage of cells in   >>>each pick,   >>>but then
I have to compare the percentages in each one of   >>>the 5-6
picks,   >>>which makes my point (more or less proliferation) not
easy   >>>to deliver...   >>>Should I , for example, compare only the
"non-proliferating"   >>>cells (=   >>>first pick) with all the others
together? Or only the "most   >>>proliferating cells" (= last pick)?

---------------------------------------------------------------------------------
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Received on Wed Mar 28 14:18:00 2007
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________________________________________
We are having problems with the CD8betape mAb from Beckman staining
HIV
+   >whole blood before lysis and permeabilisation and staining with
mAb

Beckman staining HIV
CD8beta stain probs
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From: Geza Paukovics <pauko...@burnet.edu.au>
Date: Thu Mar 22 2007 - 19:37:19 EDT

individuals. We observed poor   >CD8beta staining in two HIV+
patients and when exposed to light during the   >permeabilisation
step, the CD8betape mean flourescence of the positive   >population
increased with exposure to light. We repeated this experiment   >and
did not observe the poor CD8beta staining, and the mean fluorescence

Does   >anyone have any suggestions as to what may be causing this?

fanx for your time and attention...  cheers  Gez              Geza
Paukovics B.MLS.  Senior Research Assistant  The Macfarlane Burnet
Institute for Medical Research and Public Health  Site address:
Alfred Medical Research & Education Precinct (AMREP),  corner Punt &
Commercial Roads, Prahran 3181 or  GPO Box 2284, Melbourne
3001               .***.        ***  AIDS Pathogenesis Research
Unit    _ _|\
* | | | *        * | |  VHPF Flow Cytometry Program Group    /
\   *   * | | | *    * | | |  Sidney Myer Fund Flow Cytometry Unit
\_.--._/ * * | | |  *             O   *** ***  Monash Central School
of Medicine (AMREP)  Flow-Cytometry and Sorting Suite Co-ordinator
tel: (+61 3) 9282 2246 (Burnet) or 9903-0601 (Monash)  fax:(+61 3)
9282 2100  email: pauko...@burnet.edu.au   This attachment - 'Purdue
CD8B staining.doc' - 31.23 KBytes - can be viewed at
http://www.cyto.purdue.edu/MD-parts/009eeed9cf8a14282a5f6bfed895a218b...
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Despite of my positive experience with the FACSFlow I would not buy
it   again.the FACSFlow I would not buy it   again.     There is a
better solution for the LSR II. BD

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From: <akos.szilv...@novartis.com>
Date: Thu Mar 29 2007 - 14:20:34 EDT
Elizabeth,    I have used several of the FACSFlow systems for a long
time. We had no   problem with any of the units.
They are reliable and need practically no   maintenance.     Despite
of my positive experience with the FACSFlow I would not buy it
again.     There is a better solution for the LSR II. BD offered us 20
liters steel   tanks. This is the same tank as the regular LSRII tank
but much bigger.   Answering your other question, yes, the HTS uses a
lot of sheath fluid   because the sheath fluid serves as the wash and
rinse agent to the   sampler. This is very convenient for not having
to monitor yet an other   container's liquid level. Actually we
switched to distilled water for   simplicity and less complication
with salt deposits. In order to monitor   the water level in the tanks
we put them onto a bathroom scale. Once you   calibrate the scale
(read the weight of the empty tank and the full tanks)   you know when
to fill it up. We have not noticed any pressure instability   with low
level of water in the sheath tanks.    Akos
__________________________   Akos Szilvasi
NIBRI Core Laboratory Services  manager  USCA, 601-5301  Novartis
Institutes for BioMedical  Research, Inc.  100 Technology Square
Cambridge, MA 02139  USA  Phone: +1 617 8717177  Email :
akos.szilv...@novartis.com              "Kraus, Elizabeth"
<eliza...@BaylorHealth.edu>   03/29/2007 01:56 PM    To  Cytometry
Mailing List <cytome...@flowcyt.cyto.purdue.edu>  cc    Subject  LSR
II FACSFlow system                Hello all...    Please let me hear
from you if you have an LSR II with the FACSFlow and   HTS Systems.
My  questions are:    1) Would you purchase the FACSFlow System
again?  2) Does the HTS use more sheath than regular tubes?    For
those of you with the standard sheath system... How long does a tank
last if running  samples all day long?    Thanks in advance!
Elizabeth    Elizabeth T. Kraus  Manager, Flow Cytometry core
Facility  Baylor Institute for Immunology Research  3434 Live Oak,
Suite 200.1  Dallas,  TX.  75204  214-820-3586
eliza...@bhcs.com      -----Original Message-----  From: Julie
Bertout [mailto:julie.bert...@ibl.fr]  Sent: Wednesday, March 28, 2007
8:58 AM  To: cyto-inbox  Subject: lymphocyte cell cycle analysis
Hello,    Does anybody have a protocol to study cell cycle of
lymphocytes purified  from blood?    I suggested the regular PI
staining I used for cell line  - fixation with cold EtOH 80%,  -
storage at 40C,  - stain 1 million cells with 100ug IP and 125ug RNase
30min @ 370C  but the morphology of the cells completely changed after
this treatment.  The lymphocyte were quiescent. The researcher did a
positive control  using PHA to induce cell cycle but the populations
changed even more...    Thank you for your advices.    Julie Bertout
cytometry lab  Institut Pasteur de Lille  1 rue du professeur
Calmette  59800 Lille  France    This e-mail, facsimile, or letter and
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Received on Fri Mar 30 12:18:00 2007
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___________________________________________Re: Unstable FSC on
FACSCalibur
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From: Randy T. Fischer <fisch...@mail.nih.gov>
Date: Fri Mar 16 2007 - 09:34:40 EDT

Hi Carl,    In the past when we have seen a similar problem it was due
to small clogs in  the fluidics.  Unfortunately, these 3micro clogs2
can take some extra effort  to clean out of the system if they are in
sample line. So, the first step  is to make sure the system is clean,
then QC it (whatever is your  routine/daily procedure for QC). Next,
assuming the instrument passes QC as  this would indicate a sample
problem and not an instrument problem, have  your users/operators
filter the samples right before they put them on the  instrument.
Depending on the cell type, they can begin clumping within a  matter
of minutes.    Good luck,    Randy T. Fischer, NIH/NIAMS  B Cell
Biology Group  9000 Rockville Pike  Bldg 10, Room 6D50  Bethesda, MD
20892  (301) 594-3537 (voice)  (301) 402-2209 (fax)        From: Carl
Simard <Carl.Sim...@hema-quebec.qc.ca>  Date: Thu, 15 Mar 2007
10:53:22 -0400  To: cyto-inbox  Conversation: Unstable FSC on

FACSCalibur  Subject: Unstable FSC on FACSCalibur

We sometime have a problem with an unstable FSC on our FACSCalibur.
By  unstable, I mean that the FSC change by steps over time. When
making a FSC  vs time plot,  you expect to have a regular line. The
line we get is a  broken one, with shifts going up or down erratically
over time. Any ideas to  what may cause these FSC shifts ? Is this a
problem with the samples or with  the FACS ?

Thanks,  Carl
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I HOPE THIS GETS TO THE PROPER PEOPLE LIKE YOU REQUESTED ON THE PURDUE
CYTOMETRY MAIL LIST

Fwd: Would you please forward my question to the Purdue cytometry
maillist?
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From: Lora <lbar...@chla.usc.edu>
Date: Mon Nov 20 2006 - 13:05:21 EST

Is it possible to help out this person?    Begin forwarded message:

2006 1:22:54 PM PST  > To: lbar...@chla.usc.edu  > Subject: Would you
please forward my question to the Purdue    > cytometry maillist?  >

subscribe to the Purdue    > Flow Cytometry Email list due to some
email problems.  Would you    > please forward my question to the
email list?  Thank you very much!  >  > Sincerely,  > Xinxiang  >
~~~~~~~~~~~~~~~~~~~~~~~~  > Hello,  >  > I recently encountered a
weird phonomenon.  After staining of yeast    > spheroplast, only 3%
of the cells were positive in terms of cell    > surface protein
expression, as determined by  BD LSRII.  But 30%    > were positive
when the exact samples were run on Guava EasyCyte    > Plus!  I wonder
what causes such great difference between two flow    > cytometers and
which machine I should trust. My instrument    > settinges seeminly
were correct because both machines
detected both    > positive and negative cells. The difference is the
fraction of    > positive/negative population.  >  > Please email me
directly at wan...@medimmune.com if you have any    > hints.  Thanks!

******************************************  > Scientist I  > MedImmune
Inc.  > One Medimmune Way,  > Gaithersburg, MD, 20878  >  >
Wan...@Medimmune.com  > Tel: (301)398-4517
Received on Tue Nov 21 13:18:00 2006
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