Detecting b-gal in cultured cells ?

[Martin Kennedy]

In article <simmenlab.33.2DF78727 at animal.ufl.edu>, simmenlab at animal.ufl.edu writes:
>  I am transfecting cells in culture with a b-gal expression 
> vector.  I was wondering if it was possible to detect b-gal expression without 
> preparing cell extracts.  I just want to know if the transfection worked or 
> did not work.  If anyone has any experience with this please let me know.  
> Thank you very much!

>                                                           Karen Reed


Karen, here is what I do:

Staining mammalian cells with X-gal


Reference:	Allen, N.D. et al. (1988).  Transgenes as probes for active 
chromosomal domains in mouse development.  Nature 333, 852-855.


1.	Aspirate medium off adherent cells, or harvest suspension cells by 
centrifugation.

2.	Rinse twice in PBS.

3.	Fix for 5 minutes (see below)

4.	Rinse twice in PBS

5.	Add stain, and incubate at 37 oC.  Blue colour may be visible within 
20 minutes, but can stain overnight or several days without causing harm or 
background problems.


Fix:		
To 100ml PBS add:	2.5ml 40% formaldehyde
			0.4ml 50% glutaraldehyde
Stores indefinitely at 4 oC.


Stain:		Solution 1:
			88ml PBS
			0.1ml of 1M  MgCl2
			2ml of 2% X-gal (made up in DMF)

			Solution 2:
			210mg potassium ferrocyanide dissolved in 5ml MPW
			165mg potassium ferricyanide dissolved in 5ml MPW
			Mix these together.

Store both solutions separately at 4 oC; keep for several months.  Before use, 
mix together solution 1 and solution 2 at 9:1 ratio (i.e. 9ml solution 1, 1ml 
solution 2).   Only make up what is needed, as this keeps no longer than 4 
weeks.

Works like a charm (I've done it on ES cells, fibroblasts and lymphoid lines)

-- 
Cheers,

Martin

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