[Cytometry] Forward Scatter Issues with BD CantoII

Jean-Pierre Aubry-Lachainaye Jean-Pierre.Aubry-Lachainaye at unige.ch
Tue Apr 16 11:43:19 EDT 2013


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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