[Cytometry] multiple negatives for comp in Diva?

mario picozza mariopicozza at hotmail.com
Fri Feb 7 20:07:05 EST 2014


I agree! 
But also have to add one point for people that compensate with cells (PBMCs in this case) and not beads. When compensating for monocyte specific markers (or markers that brightly stain monocytes such as CD14 and CD33), one has to  use the unstained monocyte population (gated in FSC/SSC plots) instead of the same tube not stained lymphocytes as the negative. The same applies to neutrophils and eosinophils (caution: this two populations broadly overlap in terms of scatter signals, but are very different in terms of autofluorescence)

Regards

mp ( :-)  )

Mario Picozza, Ph.D.
Laboratory of Applied Dermatology
Istituto Dermopatico dell'Immacolata, IDI I.R.C.C.S.
via dei Monti di Creta 104, 00167
Rome, Italy




> From: flowmail at verizon.net
> Date: Fri, 7 Feb 2014 06:44:53 -0800
> To: flowmail at verizon.net
> CC: cytometry at lists.purdue.edu
> Subject: Re: [Cytometry] multiple negatives for comp in Diva?
> 
> Oh, I see where I'm not being clear. 
> 
> The positive and negative cell/bead should match within the same tube. I was saying channel and that can be misconstrued. 
> 
> Since comp controls should be one color only, that tube (think of it as one data file) will have both the positive stained events and the denominator/baseline/control/negative events. 
> 
> Having both positives and negatives in the same tube/datafile works in all software package autocompensation wizards (Flowjo, FCSExpress, DiVa, etc).
> 
> -cbb
> 
> On Feb 7, 2014, at 6:18 AM, Cris Bare <flowmail at verizon.net> wrote:
> 
> > Nope. 
> > 
> > The only signal that matters is the fluorochrome. By matching the intrinsic signal between the positive and negative in the same channel you essentially set that intrinsic as baseline and any signal above that is from the fluor. That fluor signal is then used to calculate the slope of the spillover regression. 
> > 
> > On Feb 7, 2014, at 6:03 AM, Ian Titley <Ian.Titley at icr.ac.uk> wrote:
> > 
> >> Yep. However if Carol is using cells for the GFP channel and beads for say the PE channel will the fact that the autoflourescence of the two types of particle does not match in both channels impact the compensation between those channels?
> >> 
> >> Ian
> >> 
> >> -----Original Message-----
> >> From: Cris Bare [mailto:flowmail at verizon.net] 
> >> Sent: 07 February 2014 13:52
> >> To: Ian Titley
> >> Cc: Carol Norris; cytometry at lists.purdue.edu
> >> Subject: Re: [Cytometry] multiple negatives for comp in Diva?
> >> 
> >> Let me see of I can beat Mario this time. 
> >> 
> >> The autofluorescence ( AKA intrinsic fluorescence) should match between the positive population and the negative population within the same detector channel. 
> >> 
> >> For example, if you capture PECy7 on a bead, use the corresponding bead negative, but if you use GFP the negative should be the same cell type non-expressor. 
> >> 
> >> That's why I suggest manually drawing the "P3" negatives gate in the histogram instead of using the universal unstained negative. 
> >> 
> >> -cbb
> >> 
> >> On Feb 7, 2014, at 1:29 AM, Ian Titley <Ian.Titley at icr.ac.uk> wrote:
> >> 
> >>> Will this work if the "autofluoresence" intensity of the "negative" beads and "negative" cells are significantly different within the same compensation experiment? I think this might be what Carol is concerned about? Can Carol use antibody labelled beads as "positives" and unlabelled cells as the universal negative control within the same compensation run?
> >>> 
> >>> Best wishes
> >>> 
> >>> Ian
> >>> 
> >>> 
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