[Cytometry] Forward Scatter Issues with BD CantoII

Cris Bare flowmail at verizon.net
Tue Apr 16 13:23:31 EDT 2013


Jean-Pierre,

I disagree. While CST will warn or fail for alignment that is outside of tolerances enough to predict data trouble? It may still pass with enough misalignment to tail the FSC. 

Check the LJ plots to see if there has been a jump in blue laser cv or pmtv. This also indicates misalignment. If they are nicely flat over the same period of FSC tailing, your obscuration bar may have been moved or (rarely) your FSC diode may be failing. 

-cbb
http://www.linkedin.com/profile/view?id=28573199

On Apr 16, 2013, at 8:43 AM, Jean-Pierre Aubry-Lachainaye <Jean-Pierre.Aubry-Lachainaye at unige.ch> wrote:

> Hi Bryant
> 
> Apparently it is not a bad alignment issue, otherwise CST beads would not pass the performance check.
> 
> Did you simply try to decrease the Window Extension from 7 to 2 (as on the Aria) or even lower and to correctly set the FSC area scaling factor, having a diagonal in FSC-H vs FSH-A, with the same mean value on both parameters.
> 
> Also try to clean your flow cell with Contrad 15% diluted in DI water, doing a "clean flow cell".
> 
> 
> Regards
> 
> 
> Jean-Pierre Aubry, PhD
> 
> Flow cytometry facility-University of Medicine Geneva
> 1 rue Michel Servet
> 1211 Geneva, 4
> Switzerland
> 
> 
> 
> 
> 
> -----Message d'origine-----
> De : cytometry-bounces at lists.purdue.edu [mailto:cytometry-bounces at lists.purdue.edu] De la part de Bryant Hanks
> Envoyé : lundi 15 avril 2013 19:16
> À : Cytometry at lists.purdue.edu
> Objet : [Cytometry] Forward Scatter Issues with BD CantoII
> 
> Hi All
> 
> We've noticed a subtle yet significant change in FSC on our BD CantoII.
> For microvesicle work we use a 1.00um bead from Polysciences as a 'standard' for the upper limit of interest. 
> In the initial dot plot, (FSC-A log vs SSC-A log), we're used to seeing a fairly well defined spot of beads, with the usual doublets, debris, etc.
> Recently* we've observed this bead standard appear as an oblong smear across a full decade of FSC. A fresh prep of bead standard and titration has no affect.  The SSC of bead standard looks great with SSC-H histogram being sharp and consistent with previous runs of bead standard.
> FSC-A histogram is wide bell shaped; FSC-H peak starts sharp then slopes towards origin.
> I've changed the wet cart sheath capsule filter, replaced sheath and performed sheath exchange x2.
> Background is consistent with prior runs.
> 
> (We moved the PI's experiment over to the AriaII using same samples and completed set-up and run with no problems)
> 
> *After the CantoII fluidics motherboard fried and was replaced 26 February 2013.
> 
> CST looks great; bright bead fluorescence <3.0%CV on FITC, APC, & Pacific Blue.  No significant changes in other parameters of CST noted.
> 
> BD Call Center is less than enthusiastic in support as we have no service contract.
> 
> Any ideas as to what's going on would be greatly appreciated.
> 
> Best Regards,
> 
> Bryant M Hanks, MT(ASCP)
> Operator, College of Medicine Flow Cytometry Lab University of South Alabama Mobile, Alabama USA
> 
> 
> 
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