Re: FACS Aria Compensation

Hello Adam Palazzo,

1. If you are comfortable with manually adjusting compensation on a Calibur,
you should be able to do the same on an Aria. Automatic compensation is
an option, not a requirement. (However, it can be a great time-saver.)

2. Don't use auto-interval gates for the samples you've described. Use manual
gates. Automatic compensation may fail if your intervals do not contain enough
events, so you may have to include some "bright negatives" in your positive
interval (or "dim positives" in your negative interval). In these cases it
helps to shift the interval on the predominant population away from the
smaller population. The intervals should not overlap. ALWAYS check the
resulting compensation; do not assume that if the software succeeds the
compensation is correct. (Or you could use something like CD8 for your
compensation controls, even though it is not part of your experiment. See
"Compensation in Flow Cytometry" by Mario Roederer in "Current Protocols in
Cytometry".)

3. Automatic compensation requires one sample (with both negative and positive
populations) for each fluorochrome. You will need to add a PI tube.

4. You're using an Aria now, not a Calibur. You may want to consider
substituting something else for PI -- like TO-PRO-3 -- as a viability dye
for your FITC, PE, PE-Cy5/PerCP 3-color samples. There are many possibilities.

5. Hopefully your compensation controls exhibit the brightest fluorescence
intensity for your experiment. That is, I wouldn't use a control that
merely "bleeds into high signal" when the experiment includes, for example,
CD8 conjugated to the same fluorochrome. (Brighter controls equals better
compensation, to paraphrase Dr. Roederer from "Current Protocols".)

Of course, if there is any possibility that changing your system (antibody,
fluorochrome, protocol, laser excitation, etc.) produces samples with discrete
negative and positive populations, then this discussion would all be moot.

Good luck,
Eric


>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


Eric Van Buren, eric.vanburen@wayne.edu
Karmanos Cancer Institute and Immunology & Microbiology
Wayne State University, Detroit, Michigan, USA