[Cytometry] Compensation with liver cells

Mario Roederer roederer at drmr.com
Fri Jul 10 15:42:31 EDT 2009


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
>
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>
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