Do you get any sheath dropping back in your sample? If there
is something in the sheath, either antimicrobials or LPS or
so from the microcosm in the flow cytometer tubing that
could also account for the effect. Otherwise you should also
be able to create the stimulus prior to measuring with a
syringe.
As another anecdotal remark in that context I share one of
my experiences from my early days in flow where I lost the
pressure supply in the middle of a set of experiments (e.g.
the compressor burnt out). I hurriedly hooked up a gas
bottle to the sheath tank and carried on. Worked just to
finish the experiment, but when I wanted to do some more
measurements a bit later I got rather funny effects every
time I took the sample off. Only when shutting down the
instrument in frustration I noticed that I had 'fizzy
sheath' that had degassed every time I purged the sample in
the old Ortho. In the panic I had not considered the effect
of pressurised CO2 on the sheath.
Looking forward to the explanation of your stimulating
problem.
Gerhard
______________________________ Reply Separator _________________________________
Subject: macrophages/calcium
Author: RRABIN@niaid.nih.gov at INTERNET
Date: 18/05/1999 21:39
I want to thank all those that responded to my query about macrophages
fluxing calcium in response to pressure changes about a month ago.
I got many answers. Most respondants suggested that the machine is dirty.
We know that not to be true, and the fact that the flux only occurs after
pulling the macrophages off the machine adn putting it back on suggests that
it is physical stimulation. When we used the standard Vantage collection
apparatus, we decreased the calcium flux, but did not abrogate it. We
tested this by putting the Vantage pressure tubing through the time zero
apparatus, and that improved but did not abrogate the problem. We also
improved the calcium flux by turning down the sample pressure, but could not
abrogate the problem.
We actually have realized that the problem began when we replaced the top
part of the housing of the Time Zero apparatus because a small crack had
gotten to large. In retrospect this crack was probably dampening the
pressure changes and allowed us to get good data. We are going to try some
pressure sensitive valves in line to see if we can "reproduce" the crack.
Dr. Tarnok suggested the time-window method published in Cytometry, and we
may go to that. We are also going to try it on a FACScan with Fura-red and
fluo-3 because the pressure of the fluidics is so much lower.
Thank you for your suggestions.
ron
Ronald L. Rabin, M.D.
Clinical Associate
Cytokine Biology Unit, Laboratory of Clinical Investigation
National Institute of Allergy and Infectious Diseases
National Institutes of Health
Bldg. 10/Rm. 11N228
10 Center Drive MSC 1888
Bethesda, MD 20892-1888
Phone: (301) 402-4910
FAX: (301) 402-0627
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