[Cytometry] Compensation with liver cells

Ian Dimmick ian.dimmick at newcastle.ac.uk
Fri Jul 10 13:57:10 EDT 2009


We use compensation beads almost exclusively for stem cells and mature differentiated cell phenotyping. This works well if you do a number of very common sense things , 1- base your experiment settings on the cells under investigation, bead autofluorescence will vary from laser to laser and also from bead manufacturers therefore setting voltages on beads is a waste of time, the voltages you will have to change are for forward and side scatter which are of no consequence in calculating compensation 2- as mr states the compensation is as precise as is the bead fluorescent intensity, you hope that the bead intensity is in excess of the cellular intensity, if not then go back and re confirm your compensation for that parameter with your cells, however it is most unlikely that in a multicolour experiment all cellular antigens will give very bright signals , hence the continued (and successful ) use of antibody capture beads.3- I have never experienced under any circumstances where the bead fluorescence is off scale when using  the bi exponential scaling .4 I see no point in using beads that are similar in autofluorescence to the cells under test, using inverted matrix compensation autofluorescence is normalised and is of no significance.

don't confuse an issue that is pretty straight forward we have politicians for that !!

Ian 


Ian Dimmick
Flow Cytometry Core Facility Manager
Institute of Human Genetics
Bioscience Centre
International Centre for life
Newcastle Upon Tyne
NE1 3BZ
UK
Ian.Dimmick at ncl.ac.uk
Tel 0044 191 2418831
Fax 0044 191 2418666
(mob) 0044 7970344823
http://www.ncl.ac.uk/ihg
________________________________________
From: cytometry-bounces at lists.purdue.edu [cytometry-bounces at lists.purdue.edu] On Behalf Of SIMON MONARD [smonard at staffmail.ed.ac.uk]
Sent: 10 July 2009 17:48
To: cytometry at lists.purdue.edu
Subject: Re: [Cytometry] Compensation with liver cells

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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> Cytometry at lists.purdue.edu
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>
>



Simon Monard
FACS Facility Manager
MRC Centre for Regenerative Medicine
Institute for Stem Cell Research
University of Edinburgh
Roger Land Building
West Mains Road
Edinburgh
EH9 3JQ

Tel. Lab 0131 6505876
Tel Office 0131 6517265

--
The University of Edinburgh is a charitable body, registered in
Scotland, with registration number SC005336.


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