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We are attempting to flux neutrophils through a very low density receptor.
This is to confirm experiments performed in another laboratory. They did
the fluxes on a Perkin-Elmer MPF-66 spectrofluorimeter with PELCS III software
and got the following results:
Starting calcium concentration was 106 nM, and max (with this stimulus) was
199 nM. The flux was immediate, sustained for 100 seconds, and then declined
over the next 140 sec.
We have performed this experiment twice on an Epics Elite flow cytometer,
using 100 mW of multiline UV excitation from an Inova 300 laser. Short wave-
lengths are measured with a 405 bandpass filter, long with a combination 550 dL,
and 350 lp. These are our standard calcium flux arrangements. We know the
cells are being loaded, iono works great.
Our questions are:
1. How does the sensitivity of a spectrofluorimeter compare to a flow cytometer
for calcium flux. We know we will miss the first 30 seconds of action and the
spec does have an advantage there, but is it more sensitive?
2. We know that the concentration of stimulus needs to be higher than saturating
conditions, but should the necessary concentration vary between spec and flow?
(We have tried a range between saturating and supersaturated. Should we try
under saturated?)
3. We are using fresh neuts. They used neuts from the Red Cross. Potential
problem? (Always potential, but is it a real concern)
4. The spec fluxes did not involve cross-linking the stimulus. Should we try
cross-linking the ligand?
Any other helpful suggestions would be greatly appreciated.
Kris Weber
University of Michigan
(313)-747-3216
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