Detecting b-gal in cultured cells ?
mkennedy at chmeds.ac.nz
Sun Jun 12 21:30:54 EST 1994
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
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
To 100ml PBS add: 2.5ml 40% formaldehyde
0.4ml 50% glutaraldehyde
Stores indefinitely at 4 oC.
Stain: Solution 1:
0.1ml of 1M MgCl2
2ml of 2% X-gal (made up in DMF)
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
Works like a charm (I've done it on ES cells, fibroblasts and lymphoid lines)
NNNN NN Martin A Kennedy (E-mail = mkennedy at chmeds.ac.nz) ZZZZZZZ
NN NN NN Cytogenetic and Molecular Oncology Unit ZZZ
NN NN NN Christchurch School of Medicine ZZZ
NN NNNN Christchurch, New Zealand ZZZZZZZ
Phone (64-3)364-0880 Fax (64-3)364-0750
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