RE: "Fl1 drift over time"

I would first look at fluidics.

 

A clog in the lines would slow up the sample flow, leaving your cells (or beads) in the beam and detectors for a longer period and allow for larger integration of emitted light (FC-500 measures integrated signals) over time.

Run your sample using a 1-2 minute time stop and check the data acquisition rate over that period.  Does the data acquisition rate slow during that time?  If so, you have a partial clog and that is causing your upward drift of FL1 signal.

 

Phil Marder

-------- Original Message --------
Subject: "Fl1 drift over time"
From: Katrina Ann Walsh <kawalsh@unimelb.edu.au>
Date: Tue, May 08, 2007 10:15 pm
To: Cytometry Mailing List <cytometry@flowcyt.cyto.purdue.edu>

 

Dear Flowers,

 

We have encountered a perplexing problem with our flow cytometer, a FC500.

We have noticed that the fluorescence intensity of back ground controls, such as isotype controls or unlabelled cells appear to increase rapidly over time, in the order of doubling every 20 seconds. This is quite troubling when setting negative background, especially when positive staining is a shoulder rather than a distinct population.

We notice that it occurs on fl1 and subsequently fl2, however it is not obvious on fl4. We are told our 488 laser is behaving correctly.

We have tested epithelial cell line, lymphocytes and cyto-comp (Beckman Coulter) lymphocytes and find large cells, but NOT small beads, exhibit this "fl1 drift" over time.

We have also tested buffer ph and resuspending cells in isoton. We find that MFI fluctuates when the same samples are re-read and that this problem occurs most often in the evening, but not always.

Often we do not seem to have a problem at all.  The flow cytometer room is air-conditioned  reasonably well and would not get over 27 during the summer.

What could be the problem? All suggestions welcome.

 

Thank you

 

Katrina Walsh

School of Dental Science

University of Melbourne.

email: kawalsh@unimelb.edu.au