Thank you to all FlowPeople who responded to my requests for further info on
several subjects. This is a wonderful user's group--so many people I don't
know took the time to send me a bite of information, which, when coupled to
all the other bites became a real meal. I even got one reply asking me for
a date (I wish I were young). Interestingly, many flowpeople were
interested in the exact same questions I posed, and asked me to send them a
summary of the responses. Here goes:
re: Mouse book. No one had any suggestions for a book or collection
of articles specifically designed for flow cytometry studies in mice.
Certain chapters in Current Protocols in Cytometry and in Current Protocols
in Immunology were helpful. e.g.although not flow, for Delayed Type
Hypersensitivity measurements see Curr Protocols in Immunology pp4.5.1 to
4.5.5.
re: Splenocyte numbers. Various flowpeople reported to me their
range of recovered splenocytes obtained from the spleen of a single mouse
was 30 to 80 milllion. With a mean of about 50M. Therefore, a single mouse
can provide sufficient numbers of splenocytes for several flow cytometric
assays, which answers my IACUC mouse number requirements.
re: Portable cytometry. With all the replies, it seems an awful lot
of people are interested in studying stress in the jungle. This institute
(US Army Research Institute for Environmental Medicine) is tasked to provide
new agents and procedures to help soldiers subjected to prolonged heat,
cold, hypoxia (mountains), sleep-deprivation under-nutrition and
psychological factors. We have the capability to carry out those studies
under laboratory conditions in various environmental chambers simultaneously
cardiovascular variables and blood chemistry, and out in the field in
jungle, desert or on mountain-top (Pike's Peak) on soldiers and military
recruits taking physically and mentally demanding courses. The goal is to
improve performance (speed, distance, mental acuity) under stressful
environmental conditions. To that end, it would be helpful to have access
to a light-weight, portable, and very rugged flow cytometer (albeit with
limited functions) that can be carried along, to better study alterations in
the immune system due to stress. RE: USARIEM Mission-- If anyone has
suggestions and/or very strong interest in studying the effects of various
stressors on human immune parameters, please feel free to contact me.
Not surprisingly, several people said they don't know where there is
one, but if there is one in the world, then Howard Shapiro would know, and
if he doesn't know about it, he can build me one. Well, after chatting with
Shapiro, it's clear that as a weekend project he could build one out of the
spare parts and pieces cannibalized from old cytometers and carwrecks just
lying around his lab and still have time to compose a few more songs.
Robb Habbersett mentioned an experimental instrument developed at
Los Alamos,where is located The National Flow Cytometry Resource. Ongoing
projects there include the development of Phase Sensitive detection schemes
which respond to the lifetime of fluorescence emission and not just spectral
properties. Also, they have several flow instruments - not strictly
cytometers - that can
measure the size of DNA fragments in flow. Habbersett was working on a
compact instrument (which could be made portable) that can "see" individual
phycoerythrin molecules in solution or detect DNA fragments (individually)
ranging in size from 125 bp to > 350 kbp. It uses a small solid-state laser
and a solid-state photon-counting detector. More information can be
obtained from the National Flow Cytometry Resource - among other things they
make their instruments and expertise available to investigators around the
country (as well as the world sometimes) - to do experiments they can't do
on their own instruments. They build instruments, data acquisition systems,
etc. Gary Salzman developed a portable flow cytometer working in Life
Sciences for many years, until recently. Howard Shapiro goes on to say,
"The machine Gary Salzman built cost the Army $6.5 million; it was intended
to replace a Coulter XL, and is smaller, but only by half. It was designed
to be mounted in the back of a Humvee equipped with air concentrators and
various other equipment.
Robb Habbersettthers suggested a device from Aber Instruments and
Optoflow with the following Stated specifications: 5mW diode laser, 635nm,
one avalanche photodiode scatter detector, one avalanche photodiode
fluorescence detector (650 - 900 nm), Battery pack:Internal 12 V, 2.5 Ah,
rechargeable NiCd battery for 2-3 hours use. The companies can be reached
at http://www.optoflow.com and http://www.aber-instruments.co.uk/. One
person informed me that Optoflow has 3 distributors in the US.
Furthermore, Oddbjorn Gjelsnes "started the company (in Norway). Nice
fellow, had worked for Skatron
when they were marketing what became the Bio-Rad Bryte cytometer."
Partec Co. informed me to go to this website http://www.partec.de
for some information about the Partec PA Ploidy Analyzer and the CCA Cell
Counter Analyzer, which are quite compact and worldwide used for routine
cell analysis.
And then there is the CytoBuoy (http://www.cytobuoy.com). The cytometer
module is a 38 x 48 cm cylinder that goes into the small 90 cm Datawell
Waverider buoy for the in situ measurement of various marine organisms.
BD responded with: The BD FACSCOunt was designed for field use,
however, it is a bit heavy to be "portable". It is a flow cytometer with two
fluorescence parameters and one size parameter. The present commercial
version is programmed to perform leukocyte CD3 CD4 and CD8 subset analysis.
re: CD/CD8 ratio increasing from 1.74 top 2.39. Is it serious?:
Very few responses were received.
Simm Maciej notes that when the ratio becomes lower than 1 it is
used to assess well-being of their HIV patients.
Phil Barren replies "it is small and unimportant, but represents the
dynamics of a biologtically intereactive system."
Randall Smith notes that in a typical hematology text a 1:1 or 1.5:1
ratio is the center of the normal range and suggesting that ratios elevated
to 3:1 during infection will return to normal over time. This suggests that
the above "...ratio of 2.39: 1 is not remarkable....". He also refers to
interesting observations of the ratios in Chronic Fatigue Syndrome patients
taken over several years.
Dennis Sasaki reported that femals generally had higher CD4/CD8 than
males, and the ratio increased with age. (Lifson et al. Clin Immun
Immunopath. 1985, 36:151.)
Re: Method for determining Th1/Th2 Ratio: There were many responses
to this very hot topic: In fact, it appears that Mike Koratich at Southern
Research Institute does it for breakfast.
Calman Prussin wrote " Perhaps this is an oversimplification but
here goes: ELISA measures the bulk production of cytokines whereas cytokine
flow measures production at the single cell level. Typically one's results
demonstrate production of both IFN and IL-4. If you note a huge
predominance of one cytokine or the other, then you very like have a Th1 or
Th2 polarized immune response. Where I see many researchers fall down is
when they find both IFN and IL-4 in a culture supernatant (by ELISA) and
attribute this to a Th0 response. In fact these investigators do not know if
they have measured a true Th0 response or concurrent Th1 and Th2 responses.
The whole Th1/Th2 paradigm rests on T cell clonality. That is, elevated IFNg
AND IL4 could mean that 1) there are ONLY Th0's, OR 2) it can mean that the
cell population contains similar amounts of Th1 and Th2 plus Th0's.
There was agreement that for ease, convenience, speed,
reproducibility and sensitivity, ELISA is the better method. However, ELISA
can not analyze the cell phenotype responsible for the cytokine production.
Flow will quantify how many of the T cells actually produce the specific
cytokines. ("Check recent publications by Kim Bottomly and many others.")
Sundararajan Jayaraman notes that Human Th1 cells produce IL2 and
IFNg but no IL4; Human Th2 cells produce IL4 and some IL2; but Mouse Th2
cells produce IL5, but no IL2. Suggests using an ELISA to look for the Th1
pattern (IL2 and IFNg) and Th2-0 like pattern (IL4 and IL5) of cytokine
secretion after stimulating PBMCs with PMA (50-100ng/ml) and ionomycin
(1ug/ml) per million PBMC in RPMI. For IL2, assay 24 hr supernatant and for
IFNg assay at 48 hr.
Meetings: Mentioned were: the 20th ISAC conference, Montpellier,
France, 20-25 May. It is the premiere meeting for flow cytometry and the
ISAC Society. You can get details and abstracts (probably) from the ISAC
web site or e-mail isac@isac-net.org
Several former students reported as did Mary G. "There is a great
flow cytometry course held at Bowdoin College in Brunswick, ME in June. I
went 2 years ago and found it interesting and challanging. You can find out
more about the course by going to www.vsh.com. Actually, I sent my
technician there 4 years ago and still raves about it, so I registered to
take it, myself, this June.
Chicago Clinical Flow and Image Analysis Course. Aug 4-11, 2000.
Northwestern University,Chicago Contact Dr. Charles Goolsby
c-goolsby@nwu.edu
It's too late to go to this one--Kananaskis Tumor Heterogeneity
Symposium (somewhat like a Gorgon Conf on flow and image in human
malignancies) Feb 18-21, 2000. Kananaskis, Alberta, Canada (see
http://129.22.79.127/K4Program/Program.htm
I was informed to check out this webpage:
http://www1.shore.net/~bugbytes/. It is the boston area cytometry users
group that meets a few times a year in this area.
Thank you all,
Steve
***************************************************************
** Stephen L. Gaffin, Ph.D. **
** Research Physiologist **
** Military Nutrition & Biochemistry Div **
**US Army Research Inst for Environmental Medicine **
** Natick MA 01760-5007 USA **
** **
** Tel. 508-233-4867 Fax 508-233-4869 **
** Stephen.Gaffin@na.amedd.army.mil
<mailto:Stephen.Gaffin@na.amedd.army.mil> **
***************************************************************
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