Fixed cells - washing out fixative before running?
smonard at ibmc.up.pt
Wed Sep 26 07:03:52 EDT 2007
Thats a ton of fixative mate, why so much? Most folks use about 1% formaldehyde
for flow. I buy the good stuff that comes in glass vials for EM 16% or so. You
can make it up from the paraformaldehyde powder if you want to save money. 10%
is probably good for pickling cats, rats, lungs etc, etc. Also, check the
bottle for precipiate, That could clog your machine, if when you shake it it
look like a snowstorm chuck it out, in fact I'd chuck out anyway. If the
fixaive is old it can produce dramatic autofluorescence problems as well (
maybe its not true autofluorescence). You can indeed wash the fixative out
after a day and the cells are still good for days or even weeks, we used to do
that to reduce the autofluoresence problems.
What sort of cells are you dealing with?
Simon Quoting Adrian Smith <a.smith at centenary.usyd.edu.au>:
> 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?
> Adrian Smith
> Centenary Institute, Sydney, Australia
Cytometry Lab Manager
Rua do Campo Alegre, 823,
4150-180 Porto - Portugal
Tel +351 226 074 900 ext 3102 . Fax +351 226 099 157
Cellphone # 919 036 680
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