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Anyway, I can now smile and say thanks to everyone who made suggestions.
The major response was that the 505nm filter was too high. The day after
the machine went out of warranty, we took a dichroic from the spares box,
prised it off its holder and replaced it with a 435LP filter from Omega.
So now we were collecting the 395nm fluorescence in FL5 and the 530nm in
FL4. I must admit Im having a bit of trouble getting used to this, it
seems the wrong way round logically, anyway.......
The next problem was that BD in their infinite wisdom supplied us with
only one pulse processor board. I tried to get a ratio signal on the
second laser for two days before the idea came to me to take the card out
of slot 18 and try it in slot 17. Voila!!
Today I came across what happens if you dont rinse tubing between samples
and found that even this aspect was covered in my replies, thanks to
Dennis Young.
You know, sometimes when you read through these things, you feel you've
been really stupid. Still, it works now!
So, about making the calcium data quantitative............
Graham
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