[Cytometry] Compensation with liver cells

SIMON MONARD smonard at staffmail.ed.ac.uk
Fri Jul 10 12:48:16 EDT 2009


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!


  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

Tel. Lab 0131 6505876
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