X-gal Staining Blues -- Why Ain't My Cells Gettin' Blue?

Andres Ferber andres at calvin.jci.tju.edu
Wed Dec 1 14:05:25 EST 1993


In article <CHC17A.792 at ncifcrf.gov> masuda at fcs280b.ncifcrf.gov (Michiaki Masuda) writes:
>From: masuda at fcs280b.ncifcrf.gov (Michiaki Masuda)
>Subject: X-gal Staining Blues -- Why Ain't My Cells Gettin' Blue?
>Date: Wed, 1 Dec 1993 01:23:33 GMT
>    I'm trying to stain NIH3T3 cells which are transfected with a plasmid
>containing the lacZ gene (derived from pCH110) under the control of a
>retroviral LTR.
>    I seeded the cells in a 60 mm culture dish and tried to stain the cells
>by the following method:
>
>[1] Rinse the cells with cold PBS twice (4 ml each time).
>[2] Fix the cells with 3 ml of 2 % formaldehyde / 0.2 % glutaraldehyde
>    in PBS for 15 min at 4 C.
>[3] Rinse the cells with cold PBS twice.
>[4] Add 3 ml of PBS containing 5 mM potassium ferricyanide /
>    5 mM potassium ferrocyanide / 2 mM MgCl2 / 1 mg/ml X-gal.
>[5] Incubate the culture dish at 37 C overnight.
>
>   Essentially, I didn't see any difference between the transfected NIH3T3
>cells and the untransfected control. Although the possibility cannot be
>excluded completely that there is something wrong with the plasmid or
>the transfection procedure, results of other experiments sugget that the
>staining procedure is the prime suspect.
>
>   I'd appreciate it if someone could provide me with his/her protocol
>working successfully and/or some secret tips to help me out from...
>
>   X-gal staining blues...
>   X-gal staining blues...
>   X-gaaaal staaaaaiiiining bluuuuuues...
>
>   Thank you.
>
>--Michiaki
>=============================================================================
>Michiaki Masuda
>Laboratory of Molecular Oncology
>National Cancer Institute
>Frederick, MD 21702-1201
>E-mail: Masuda at ncifcrf.gov

I am staining mouse fibroblasts transiently transfected with a plasmid
containing the beta galactosidase cDNA under the control of the SV40 early
promoter.
It works fine for me.The conditions are those suggested by Promega
FIX for 15 minutes at room temperature in
0.25 glutaraldehyde
0.1M sodium phosphate, pH 7.0
1mM MgCl2

Stain at 37 C O/N. If I do not see anything after 2 hours usually nothing
shows even after 24 hs
The solution is
0.2% Xgal in DMF
1mM MgCl2
150 mM NaCl
3.3 mM K4Fe(CN)6
3.3 mM K3Fe(CN)6
60 mM Na2HPO4
40 mM NaH2PO4

I always wash the cells with Hank's.Twice before fixing and 3 times after
fixing to be sure that I remove the glutaraldehyde.

Most of the time I get a very dark precipitate (blue) other times the color
is somewhat pale but definitively blue.

In your case I will check the plasmid. You can also increase the amount of
x-gal from 1 to 2mg/ml.I assume that the x-gal solution is fresh.
Check also the pH of your buffers (specially the staining solution) since
the bacterial beta-galactosidase works at pH 7 and not lower.

You could also isolate some RNA from the transfected cells and do a Northern
or rtPCR to be sure that the transfection is working

Good luck
Andres
andres at calvin.jci.tju.edu



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