[Cytometry] Compensation with liver cells

Ian Dimmick ian.dimmick at newcastle.ac.uk
Sat Jul 11 13:34:49 EDT 2009

Beads can not go off scale , the relative negative events will allways be visualisable to whatever degree, it has been my experience that you can always compensate on the calibur using these beads , you would stop your compensation when the median of the bright was the same as the negative , or if you wish you could import the Calibur data into diva and use the bi exponential scale to visualise a little better the negative compensation beads , although still using the median.I did not ignore Simon's comment regarding the autofluorescence , if you re read my mail I stated that autofluorescence is taken into account in the inverted matrix calculation , whatever value it has , you defend Simon but I do not feel his position needs to be defended , we all have our ways of doing things and we all have our views on "the best" way to do things, this is why this forum thrives, in many scenarios we are all helped by people offering their comments on personal experience , and very often those views will differ , primarily because flow cytometry ,cells , filters , lasers....................are not perfect , and very far from standard, sometimes even within the same instrument platform , so please if you want to make comments then make them in context of your own experience , do not miss quote, I felt that the whole discussion was starting to have the effect of confusing rather than helping , I did not aim that at Simon , but the whole thread in general , however if Simon felt that my comment was aimed at his mail the I appolgise to him .
I do however stand by my comments 

Ian Dimmick
Flow Cytometry Core Facility Manager
Institute of Human Genetics
Bioscience Centre
International Centre for life
Newcastle Upon Tyne
Ian.Dimmick at ncl.ac.uk
Tel 0044 191 2418831
Fax 0044 191 2418666
(mob) 0044 7970344823
From: cytometry-bounces at lists.purdue.edu [cytometry-bounces at lists.purdue.edu] On Behalf Of Ray Hicks [rhicks at cytekdev.com]
Sent: 11 July 2009 16:07
To: 'Mario Roederer'; cytometry at lists.purdue.edu
Subject: Re: [Cytometry] Compensation with liver cells

So Mario and Ian,

On an analogue machine, say a Calibur, if you wish to set compensation for
bright cells, you'd set your PMT voltages so that the negative cells would
be on scale, then you pop on your compensation beads, the unlabelled comp
beads are off scale or clipped to the origin of the graph, the stained comp
beads are on scale, you start compensating them so that the spillover
fluorescence is equivalent to that on the unstained bead, you hit a pitfall,
in that you don't know when to stop.  Whether you use mean or median or
magic, you're not going to know when you've applied enough compensation,
since you don't know where the negatives lie.  Using compensation beads that
have enough "Autofluorescence" to be on scale at the same time as the
stained ones is pretty much essential I'd say under this circumstance - one
that Simon described and which you've both ignored in your answers.

Care would probably have to be taken that the bead autofluorescence was
neutral over the wavelengths investigated.

I don't think Simon was confusing any _issues_ Ian


Ray Hicks


Tel: +44 (0)208 1337 968  Fax: +44(0)208 5889 004  Skype: ray.hicks.cytek

Cytek Development Europe, Unit 8 The Maltings, Millfield, Cottenham,
Cambridge CB24 8RE. UK

-----Original Message-----
From: cytometry-bounces at lists.purdue.edu
[mailto:cytometry-bounces at lists.purdue.edu] On Behalf Of Mario Roederer
Sent: 10 July 2009 20:43
To: cytometry at lists.purdue.edu
Subject: Re: [Cytometry] Compensation with liver cells

As I wrote, as long as your positively-stained beads are at least as
bright as your stained cells, they are a perfectly good compensation
control.  In today's instrumentation, it's irrelevant if the negative
(unstained) beads are "not onscale".  With modern digital electronics,
it's not even possible, since all signals are collected in linear, for
which even zero measured fluorescence is still onscale.

So in fact, the background fluorescence of compensation beads is not
relevant, and there is no need to match it to the cells. Don't worry
if the beads are pegged at the bottom when you run on the voltages
optimized for your cells.

So.... In summary:  No!


On Jul 10, 2009, at 12:48 PM, SIMON MONARD wrote:

> No...?
> If you are looking at larger cells, ESCs, Macs, hepatocytes etc you
> will find that setting your voltages on the negative cells the beads
> will be offscreen. Bump up the voltages enough to get the beads on
> screen and you may find you have little dynamic range left to measure
> fluorescence. You can find that your negative cells are two logs
> higher than blank comp beads. If you have an older machine with 4 log
> decades BD comp beads are useless if you work with such cells. The BD
> beads have clearly been selected because they have a similar
> background to lymphocytes. Larger beads have a higher background but
> also capture more antibody so they can be a lot brighter than the
> commercial beads. I'm not suggesting adding background fluorescence to
> blank beads I'm suggesting selecting beads that better match your
> cells and coupling them to anti-rat Ig or whatever. 6 micron
> polycarbonate beads are a much better choice for larger cells.
> In summary...Yes!
> Simon
>  Quoting Mario Roederer <roederer at drmr.com>:
>> No... antibody capture beads are not designed with a particular cell
>> subset in mind.  Any compensation bead will work for any cell type,
>> as
>> long as the positive beads are brighter (or at least as bright) as
>> your stained cells.
>> Adding background to your beads (or using beads with more background)
>> will only decrease the precision (although not the accuracy) of your
>> compensation.
>> mr
>> On Jul 10, 2009, at 5:24 AM, SIMON MONARD wrote:
>>> You will likely find them unusable as the commercial ones are
>>> designed
>>> for use with lymphocytes. If you are planning on doing much FACS
>>> then
>>> I suggest you make your own capture beads using beads with a
>>> background fluorescence comparable to your unstained cells
>> _______________________________________________
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> Simon Monard
> FACS Facility Manager
> MRC Centre for Regenerative Medicine
> Institute for Stem Cell Research
> University of Edinburgh
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> West Mains Road
> Edinburgh
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