Flow cytometry

SLForsburg forsburg at sc2.salk.edu
Fri Sep 30 10:00:17 EST 1994


In article <36fnjh$5hi at news.mic.ucla.edu>, colleen hayase
<hayase at ewald.mbi.ucla.edu> wrote:

> I prepared yeast cells (Saccharomyces cerevisae) for flow cytometry as
> follows:
> 
> 1.  Fix cells in 70% EtOH overnight at 4oC
> 2.  Resuspend cells in 50 mM sodium citrate, and  sonicate
> 3.  Add RNase A to 1mg/ml, and incubate for 1 h at 37oC
> 4.  Then I went to the flow cytometry facility to stain the cells.  The
> staining buffer they use over there was:
>                       4 mM    Sodium Citrate
>                       0.3%     Triton-X100
>                       0.1 mg/ml   Propidium iodide
>                       0.02 mg/ml   RNase A
>    The staining was about 30 min.
> And the operator then run my samples and told me that the dye did not get
> into cells!
> 
> Can anybody give me some hints?

I have done a fair amount of FACS on both cerevisiae and pombe.  I fix
them in ethanol, rehydrate them in 0.5ml 50mM citrate, RNAse them for 1-2
hours, and then stain them *overnight* by adding an equal volume of 50mM
citrate with 8ug/ml PI so that the final concentration of PI is 4ug/ml and
the final volume is 1ml.  You need to be sure you have enough cells, first
off, and that you are comparing equivalent numbers in different samples; 
1 to 5 x 10^6 is ballpark.  Your FACS operator may not realise how small
yeast are and how weak the signal is relative to larger eukaryotes if s/he
hasnt run yeast before.  For a Becton Dickinson FACScan, we start with an
FL2 voltage of about 890, and use the E00 detector for forward scatter. we
then tweak the signal with voltage and amplifier until it looks right
before doing the run.  If you have adequately fixed your cells and given
them long enough to stain, they should be fine.  

susan

-- 
forsburg at sc2.salk.edu
    formerly forsburg at molbiol.ox.ac.uk



More information about the Yeast mailing list