Fixed cells - washing out fixative before running?

Ernest Stapleton Ernest.Stapleton at easternhealth.ca
Tue Sep 25 14:15:21 EDT 2007


Hi there Adrian,
It's easy to find out, spin down a fixed sample, make a wet prep on a slide and check with a microscope. You will know right away if there is a sample problem.
If they are diluting fixative from a stock, there could be precipitates present. If this is so, they can filter the NBFormalin through .45 micron syringe filters after preparation. Obviously something in the samples is clogging your LSR, usually a direct look on a slide is all it takes to find out!
 
Cheers,
 
Ernest Stapleton 
Division Manager 
Immunology and Genetics Laboratories 
Room 1524 Health Sciences Center 
St. John's, Newfoundland 
A1B 3V6 Canada 
709-777-8654 

-----Original Message-----
From: Adrian Smith [mailto:a.smith at centenary.usyd.edu.au]
Sent: Monday, September 24, 2007 11:55 PM
To: cyto-inbox
Subject: Fixed cells - washing out fixative before running?


Hi all, 

We've been having some problems with repeated (partial)clogging on our LSR-II and are working through possible causes and solutions. 

One thing that has emerged in the past couple of days is that there is one group that runs fixed samples that are still sitting in the fixative (10% neutral buffered formalin), ie they don't wash the cells after fixing. There has been some suggestion that this might be contributing to the problem. Has anyone ever run into problems with people doing this? I've always washed cells after fixation and resuspended them in something else (like PBS) so I've not considered it before - it doesn't sound like it should create problems but I'm interested to hear from others.

Incidently, the same group is having a lot of problems running the same samples on an AMNIS Imagestream (the flow rate is unstable after the first couple of minutes) - could the presence of formalin also be an issue there? 

Regards,

Adrian Smith
Centenary Institute, Sydney, Australia




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