[Cytometry] Doublet discrimination on the Calibur

Cappella, Paolo Elia [Nervianoms] Paolo.Cappella at nervianoms.com
Fri Sep 6 11:08:13 EDT 2013



The only one way that I know to use scatter parameter (SSC) using on
FACSCalibur is unmount physically side scatter coaxial cable at exit of
PMT and connect instead a fluorescence PMT on electronical board and
deceive CellQuest on DDM (Parameter description). Of course using this
you could have a FACSCalibur with 1 less fluorescence that is
sacrified.I know that it is not perfect, but you can analyzed
fluorescence (FMO) plus SSC-H/FSC-H and SSC-H/SSC-W. (You have
experience is electric-electonic device, because it's not an OEM change
and electricity could be hazardous-for each change swith off machine!!!.
However you'll find a fantastic trick that I used in my first StarPlus
almost 18 years ago in my first lab :-; ). Joanne is perfect in her
answers, use nuclear dye (see current protocols of cytometry, PI for
cell cycle etc) . Another suggestion: you could be use the brighter
fluorescence you have in your panel, put in linear scale and push pmt
voltage. If you are lucky you could difference in FL(X) Hight vs Width
and you could get off your aggregate. Unfortunally I know a lot of cell
lines (e.g. HCT-116) that has aggregate after detaching process. Try to
wash very well your cell colture with PBS without Ca/Mg, use
tripsin/EDTA 1:10 for few minutes warm at 37, try addition enzyme (I
used in past Accutase/Accumax, sometime worked), use a acquisition
buffer containg EDTA.
Anyway good work and good luck!
Paolo



-----Original Message-----
From: cytometry-bounces at lists.purdue.edu
[mailto:cytometry-bounces at lists.purdue.edu] On Behalf Of madan verma
Sent: Thursday, September 05, 2013 1:49 AM
To: cytometry purdue
Subject: Re: [Cytometry] Doublet discrimination on the Calibur

Hi ,
Doublets can be exploited by using FSC-A(area) Vs  FSC-W(width), we have
Navios and both Parameter are  available , though you have to select
them when making protocols. Also I am not sure about BD, you might have
to play arround, this also ensures that you don't waste a FL channel.
Thank you.
Madan Verma

Hi Rob:
You only have access to doublet discrimination on your fluorescent
parameters on a FACSCalibur. Most likely the ones you have seen DD on
FSC and SSC have been on digital instruments. I suppose you could do a
nuclear stain on your cells and use that parameter doe DD, but that will
take up one of your 4 channels (two if you are using the width
parameter). You can reduce the incidence of coincidence events by
running in Low and diluting your sample. Perhaps the reviewer is not
familiar with the Calibur electronics.
Good Luck,
Joanne

 
________________________________________
 
Paolo Cappella, Dr. Biol.
Cell Assays Technologies / Flow Cytometry
Building 75 - Discovery
Nerviano Medical Sciences srl
PO Box 11 - Via Pasteur 10 
20014 Nerviano MI
phone: office +390331581367
           lab    +390331581778
           cell   +393395499154
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