RE: FACS Aria Compensation

Hi Adam.
Beads are very different to cells. In my experience sometimes you have
distinct peak and sometime a continuos fluorescence.
I'm not an expert of FACSAria, but i work on FACSVantage.

My suggestions are:
1) Exclude all auto-aligments, compensations or setup during acquisition;
try to learn how change parameters for each function using beads and CELLS!
Beads are very little than cells and more bright comparing cell staining;
2) If is possible, compare your acquisition using other FACS (FACSCalibur,
for example)
3) Use right controls (all dyes and mouse or rabbit IgG control)
4) Try to stain same number of cells
5) Try to acquire samples without compensation and use post-compensation
software
6) Semplify your procedure, planning what is tha aim of your experiment,
what is your readout, what is relevant to find (which antibodies...)
7) Compare togheter florochrome emission spectra (look where do you expect
the cross-talking for example.
These are common suggestion.

Regards Paolo Cappella
Flow Cytometry Unit
Biology Dept. - Bdg.75 
Nerviano Medical Science srl
Via Pasteaur 10 - I 20014 Nerviano
tel. +390331581367

-----Original Message-----
From: Adam Palazzo [mailto:Adam.Palazzo@UTSouthwestern.edu]
Sent: Tuesday, September 28, 2004 4:16 AM
To: cyto-inbox
Subject: FACS Aria Compensation


Hello All.

I've just recently got back from my training on the Aria and Im not
working with cells instead of beads and Im running into a lot of
questions regarding the automatic compensation.  Im very new to FACS so
pardon my naievite.  

Here is my problem.  With the Calibrite beads in training when we rain
our "stained" controls, which were calibrite beads for virtually every
fluor mixed with unlabeled beads, there were two distinct peaks in the
histogram plots for each fluor.  However, when I stain cells for my
compensation controls i dont have discreet peaks.  Most of the time I
have signal that more or less bleeds into high signal from the
non-expressors.  I have used these same compensation controls with the
same cells on the calibur so i know they work as controls.  My ultimate
question is, where do i draw the interval gates for each fluor?  In the
training and in the tutorial for autocomp they have you use the
auto-interval gate because the beads give you such tight, indivudual
peaks.	Do just do my best with my compensation controls to interval
gate whats positive and negative?  Is that going to cut it?  

Also I typically compensate for PI on the calibur because I typically do
three color staining in addition to PI.  Do I add PI to the list of
compensation tubes?  

Any help will be greatly appreciated.  Thanks everyone.

Adam Palazzo
Perlingeiro Lab
Center for Developmental Biology
University of Texas Southwestern Medical Center at Dallas
214.648.7354