[Cytometry] Compensation with liver cells

Ian Dimmick ian.dimmick at newcastle.ac.uk
Mon Jul 13 06:41:48 EDT 2009


Simon, could I ask the voltages you are using on the fluorescent detectors on the Calibur, as stated I have never come across this problem , I also have a Calibur , as well as digital instrumentation 

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 

 

-----Original Message-----
From: cytometry-bounces at lists.purdue.edu 
[mailto:cytometry-bounces at lists.purdue.edu] On Behalf Of SIMON MONARD
Sent: 13 July 2009 10:56
To: cytometry at lists.purdue.edu
Subject: Re: [Cytometry] Compensation with liver cells

Ian
All the instruments in my lab are NOT digital. Probably most 
instruments in the world are not digital. Granted you could use 
comp beads on digital machines when looking at large cells. 
However, on a Calibur, as soon as 50% of the blank beads are in 
the first channel the median will be 1. Continue to reduce the 
voltages and it will remain 1. Likewise your positive beads 
will have a median of 1 when 50% are in the first channel and 
will continue to be 1 as you increase the compensation. So, the 
compensation will apparently be correct for a wide range of PMT 
voltages and compensation settings. which ever way you 
visualize and calculate compensation this will not change the 
fact you have all those uninformative ones.
I am suggesting an inexpensive solution that works with all 
platforms that can be used with real time or software compensation.

Simon

  Quoting Ian Dimmick <ian.dimmick at newcastle.ac.uk>:

> 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 ofte!
>  n 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
> 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 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
>
> Ray Hicks
>
> http://www.cytekdev.com
>
> 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!
>
> 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
>>>
>>> _______________________________________________
>>> Cytometry mailing list
>>> Cytometry at lists.purdue.edu
>>> https://lists.purdue.edu/mailman/listinfo/cytometry
>>>
>>>
>>
>>
>>
>> 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.
>>
>>
>> _______________________________________________
>> Cytometry mailing list
>> Cytometry at lists.purdue.edu
>> https://lists.purdue.edu/mailman/listinfo/cytometry
>
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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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