Re: "Fl1 drift over time"

Hi - could it be that one of your users is inadverdently loading the machine with a dye solution that lingers and then stains up subsequent samples?  I'm not sure that that would explain "fluctuations", but it may explain why you have a ramping increase in intensity of negative controls, and the fact that beads aren't affected, and it's more likely to happen in the afternoon or at least after the machine has been used.  

On our FACSCalibur we find that residual PI from cell-cycle experiments can linger for quite some time and leap out on unsuspecting fixed samples for days to come - to the extent that we've banned use of PI from that machine so that cytokine experiments don't get ruined so often.	In our case it was only fixed cells that were affected of course, or dead cells in a non-fixed prep, and the increases were in fl2 and fl3 (blue excited orange and red) because PI was the dye being used at high concentration, I'm not sure what dye might cause a rise in FL1 and FL2 - maybe a rhodamine or fluorescein derivative? Depending on how contaminated the machine was with PI, users would see their negatives ramping while trying to set up with high levels of PI, but the staining would only be obvious on re-analysed cells at low concentrations.


Hope this helps

Ray

  ----- Original Message ----- 
  From: Katrina Ann Walsh 
  To: Cytometry Mailing List 
  Sent: Wednesday, May 09, 2007 3:15 AM
  Subject: "Fl1 drift over time"



  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