Re: Conpensation on mixed cell types

As Mario says -  but first make sure that the voltages are set so that all
of your cells are on scale and that it’s a useful part of the scale (if
you’re using beads that’ll be quite likely) , and don’t worry that the
highly autofluorescent cells are offset from the origin of the plot.
Remember that if you change your pmt settings for each cell type (as I’ve
seen done a few times), the relative level of expression of markers becomes
hard to compare between types (if you drop the voltage so that
autofluorescent cells that would appear in the second decade now appear in
the first decade of a log scale, you drop the amplification of positive
signal by a factor of ten too).  

 

It may seem obvious (or maybe it doesn’t), but “correcting” for
autofluorescence of (say) cultured lymphocytes by dropping PMT voltage so
that their negatives appear in the same place as unstimulated lymphocytes,
then comparing the MFI	due to an antigen (whichever  M you care to choose)
on unstimulated and cultured cells would make it look as though the cultured
cells had downregulated the expression of the marker when in fact you’d just
turned down the amplification.	 I’ve seen people do this and also try to
compare MHC levels on monocytes and lymphocytes  in PBMC after “correcting”
for autofluorescence too – hence the overlong rant :)

 

 

 

Ray

 

Ray Hicks

 

Cytek Development Inc

http://www.cytekdev.com

tel: +44 (0)208 1337 968

skype: ray.hicks.cytek 

 

 

 

From: Mario Roederer [mailto:roederer@drmr.com] 
Sent: Monday, February 25, 2008 9:52 PM
To: cyto-inbox
Subject: [SPAM-LOW] Re: Conpensation on mixed cell types

 

Since compensation is intrinsic to the fluorochrome and independent of
autofluroescence, it doesn't matter what kind of cells you are analyzing
when it comes to compensation.	Just set your comps based on beads (or
single-stained cells), that for each color, the positive and negative
controls have the same background (autofluorescence) -- for example,
unstained beads.  And make sure that your comp control is brighter than any
sample.

 

Now your compensation is properly set.	If you are seeing events that appear
undercompensated, then perhaps this is due to very highly-autofluorescent
cells--since autofluorescence has a different spectrum than fluorochromes,
it will tend to appear on a correlated (diagonal) line in distributions.

 

mr

 

 

On Feb 25, 2008, at 2:34 AM, Rossi Ralph wrote:





Dear All, 
	does anyone have any suggestions on how best to perform compensation
on samples with different cell types .In particular

	cell types with very different levels autofluorescence ? 

thanks 

Regards  
Ralph Rossi 
Flow Facility Manager 
Peter MacCallum Cancer Centre 
Melbourne , Australia 
Email: HYPERLINK "mailto:ralph.rossi@petermac.org"ralph.rossi@petermac.org 
Ph 96563747 , 96561955 

This email (including any attachments) may contain 
confidential and/or legally privileged information and is 
intended only to be read or used by the addressee. If you 
are not the intended addressee, any use, distribution, 
disclosure or copying of this email is strictly 
prohibited. 
Confidentiality and legal privilege attached to this email 
(including any attachments) are not waived or lost by 
reason of its mistaken delivery to you.
If you have received this email in error, please delete it 
and notify us immediately by telephone or email. Peter 
MacCallum Cancer Centre provides no guarantee that this 
transmission is free of virus or that it has not been 
intercepted or altered and will not be liable for any delay 
in its receipt.

 

 

No virus found in this incoming message.
Checked by AVG Free Edition.
Version: 7.5.516 / Virus Database: 269.21.0/1296 - Release Date: 2/24/2008
12:19 PM


No virus found in this outgoing message.
Checked by AVG Free Edition. 
Version: 7.5.516 / Virus Database: 269.21.0/1296 - Release Date: 2/24/2008
12:19 PM