RE: FACSAria Question: no signal with Red laser (Trigon)

Dear Achal, 
    To solve this problem, you can start APC-cy7 or APC with bivariate
histograms (height vs area).
Even you tried with all series of reasonable numbers, you might miss
another things.
For example, Even though you have a right number for laser delay, your
sample population is located in out of range with 'high enough' PMT
voltage
(Sometimes people only seek for the answer they want.). 
Thus, you can find this population with some tricks.
 
First, you must have good FSC vs SSC signals.
Second, you can gate previous main population into APC-cy7 or APC
histograms. (sure, you can not see anything.)
Third, you can make box (or gating box) inside APC-cy7 or APC histograms
and try to find exact number of cells, which are gated from FSC vs SSC
histogram by moving it around from corner to corner. You can see the
changes of number for the population in statistics box. 
Fourth, if you find it, try to put those cells in the diagonal line of
APC-cy7 or APC bivariate histograms (height vs area) with fine tuning.
 
Hope it helps,
 
Chang
 
% Do not forget checking other basic stuffs. (wrong beads etc...) 

	-----Original Message-----
	From: Achal Pashine [mailto:achalpashine@gmail.com] 
	Sent: Tuesday, April 05, 2005 2:55 PM
	To: Cytometry Mailing List
	Subject: FACSAria Question: no signal with Red laser (Trigon)
	
	
	Dear Flowers,
	This is Aria related question and any tips would be greatly
helpful to us (till BD guys get back to us). 
	Until recently, when we ran QC with rainbow particles, we could
see signals in Blue laser related PMTs (octagon) as well as Red lazer
related PMTs (trigon). But today when I ran QC, I do not detect any
major signal on Red related PMTs with the same batch of rainbow beads.
	We checked (and incrementally changed) laser delay from -30 to
-50 and did not get any signal. Also, changed area scaling (from .5 to
2.0 incremental changes) without any effect. (also changed sheath
pressure:low high med as well as checked instrument configuration to see
whether red related PMTs are included or not, they are).
	I phyisically checked red laser is working by using shutter
override and can see both the colors (blue and red).
	Curiously, we see some very sharp noise like signal at ~60 in
APC-cy7 as well as APC area histogram that does not move at all with
change in voltage of that PMT.
	Do you have any tips as to how to regain signal acquisition for
our problem or what can be wrong.
	Thanks in advance for your insights.
	
	-- 
	Achal Pashine
	Chiron Corporation