DAPI/Calcofluor

[SL Forsburg]

Nancy A. Markley wrote:
> 
> I've been trying to stain S.pombe cells with DAPI/Calcofluor but
> the DAPI will not stain the cells although the calcofluor will.
> The final concentration of DAPI I used was 1 ug per ml of cells.
> I incubated the DAPI/Calcofluor with the cells for 10 minutes at
> room temperature in the dark followed by two washes with PBS.
> When viewing the cells, only those cells that are damaged will
> stain with DAPI.  I was under the impression that DAPI is
> lipophilic and can enter the cells without the need for
> permeablization.  Is this not the case?  Does anybody have a
> procedure that I can use?  Thanx in advance. :)
> 
> Ameet Sengar
> assengar at acs.ucalgary.ca

pombe cells ARE permeable to DAPI, even live cells are.  You can even
swipethem off a plate and put them down on slides with DAPI in the 
mounting media, so there must be another problem here.  These are the 
protocols we use:

 method 1, DAPI in the mounting medium:
a 10x DAPI stock may be prepared and aliquoted.  This is 10ug/ml DAPI,
 10mg/ml p-phenylenediamine . (The DAPI stock is 1mg/ml in DMSO. ) 
 Take a 20ul aliquot and add 180ul  100% glycerol .  This gives a 1X 
working stock (1ug/ml DAPI, 1mg/ml p-phenylenediamine which acts as 
anti-fade, 90% glycerol) which should be kept at -20°C in the dark. 
 
	method 2, wash DAPI in and out.
Alternatively, a working stock of 0.5ug/ml of DAPI in PBS may be used
 to wash the cells for 30-60".  They are then washed free in PBS.  A 
mounting medium is made from a stock of 10mg/ml p-phenylenediamine in 
1M Tris and diluted 1:9 in 100% glycerol.  This method
  will reduce the background flare from the DAPI compared to having
it in the mounting medium, as in method 1.  .

For Calcofluor,a 1mg/ml stock is made up in 50mM sodium citrate, 100mM
 sodium phosphate pH 6.0. and stored in the dark at 4°C.  This is then
 diluted to 50ug/ml with 50% glycerol containing 0.3 mg/ml 
p-phenylenediamine and used as a 1X working stock. 

Hope this helps.

On another subject, has anyone any experience with the pombe
met3 marker?  I can't get them to grow beyond microcolonies
 on 0.3mg/ml  methionine plates.  Is there a trick, or do they just 
like more met?  (Before I pour even more plates....)

-- susan


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S L Forsburg                             
susan_forsburg at qm.salk.edu        
http://flosun.salk.edu/~forsburg
 "I don't speak for the Institute,         
 and the Institute doesnt speak for me."