[Cytometry] Compensation with liver cells

Ray Hicks rhicks at cytekdev.com
Sat Jul 11 11:07:07 EDT 2009


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

Ray Hicks
 
http://www.cytekdev.com

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

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-----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!

mr

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
> Roger Land Building
> West Mains Road
> Edinburgh
> EH9 3JQ
>
> Tel. Lab 0131 6505876
> Tel Office 0131 6517265
>
> -- 
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>
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