suggestions to calibrate FACScan

From: Uriel TK <utk1@013.net>
Date: Sun Sep 26 2004 - 11:06:13 EST
hello!
I have taken upon myself to run some calibration and performance tests on 
our institution's old and venerable FACScan. Luckily the person actually in 
charge of the FACScan and the Vantage (SE) is very nice and wants to 
cooperate. Any ideas, tips, tricks, suggestion etc are VERY welcome.

1)We'll run single-intensity beads to determine CV, which I understand also 
gives a measure of the alignment of the laser. How much CV is acceptable?

2)Using 6 peak fluorospheres from Dako, I'll perform the experiment detailed 
on HS PFC 4th ed pag 201-202 to test log am linearity: I'll put the highest 
peak at or close to the top channel, acquire, and then lower the PMT voltage 
to put the highest peak 5-10 channels lower, acquire, etc and so forth. This 
will be repeated for FL1, FL2 and FL3. I am assuming that the lower 3rd of 
the lowest decade and the upper 3rd of the highest decade will be bad, but I 
do want 3 1/2 decades of dynamic range! How much deviation from the linear 
response is acceptable? should I perform it using 256 or 1024 channels?

3)Are there any other (simple) tests to measure the FACScan's performance? 
which parts need to be tested?

4)PFC states that noise can be easily 3 mV, and once again assuming this FC 
has been neglected a bit, how can I determine the noise at the lower scale?

5)PFC says that below 300 volts the PMT starts to behave non-linearly. Given 
that these PMT are old (they seem to have been replaced since it was bought, 
but not sure when), I was thinking maybe they are not behaving well even at 
higher voltages. How can we measure their performance? Is this test needed 
or not?

6)Assuming the alignment test, the log amp test, or other tests result 
unacceptable, is there something to be done by us, or the only way is to 
call the BD technician? Part of my effort here is that in case the 
FCytometer is not performing well, show them, so that they will be compelled 
to place a maintenance call, since it has been long since it was done last 
time. You know, "if it's working, why does it need repairs?". If the tests 
are ok, even better! They do have Current protocols in FC, but I haven;t had 
the time to sit and read the relevant chapters, although I'll do it before 
getting to work.

Thanks in advance,

Uriel. 
Received on Mon Sep 27 15:18:00 2004

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