[Cytometry] PI intensity moves during analysis

Dmitri Lubenets lubenets at ut.ee
Wed May 11 06:57:57 EDT 2011


-----Original Message-----
From: cytometry-bounces at lists.purdue.edu
[mailto:cytometry-bounces at lists.purdue.edu] On Behalf Of Elisabeth
Freyer
Sent: 10 May 2011 14:35
To: Cytometry at lists.purdue.edu
Subject: [Cytometry] PI intensity moves during analysis

Hello everybody,

I am observing some strange shifts of the PI intensity when i do cell
cycle analysis. I'm not talking about the shifts between samples, my
whole histogram of the same sample actually moves up in intensity within
the first minute of running it on the machine. After a minute it stays
about the same.

I use an Aria2, measure PI in 488-685/35BP It ONLY happens when I use
protocols that require treating the cells with Pepsin or Trypsin (e.g.
Vindelov) prior to  PI staining. no movement w/ EtOH fixation and
staining in PI/RNAse solution only.
The samples are sitting on ice until they get loaded onto the machine.  
I thought it might be temperature related, but also with a cooled sample
chamber it still moves.

To record I now usually wait for a minute for the PI intenisty to
stabilize and record then. It gets a bit difficult when I have samples
with very small cell numbers :(

I'd greatly appreciate if somebody could enlighten me where that comes
from and if there is anything I can do to stop this.

Cheers
Lizzie
:)

Dear Lizzie, I have the same thing happening on our AriaI when analyzing
stem cells stained with Partec DAPI. Although cells were stained only with
DAPI the increase in fluorescence affected FITC (530/30) channel as well. It
was like auto fluorescence increases with time and after 1 minute or so goes
down. In the beginning I thought that our cytometer is broken, but nothing
similar happened with beads. When I run a probe containing mixture of cells
and beads it become clear. Signal from beads were constant all the time in
all channels while signal from cells were fluctuating. I found out that
simply diluting Partec DAPI solution 2 times with buffer prevents this
phenomenon from happen. I believe that Partec DAPI contains saponin (or
other compound affecting membranes) which does something to cells. Try to go
down with Pepsin of Trypsin concentrations. May be that helps.  
Best wishes, Dmitri Lubenets




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