MarkerGene™ in vivo lacZ β-Galactosidase Intracellular Detection Kit
Product ID: M0259
This kit allows ultra-sensitive detection of β-galactosidase activity in live mammalian, yeast, bacterial, or plant cells. It is especially useful for flow cytometry applications.
Although chromogenic assays of beta-galactosidase activity (i.e. X-Gal) have use, the recent application of the fluorogenic substrate fluorescein di-β-D-galactopyranoside (FDG) combined with fluorescence microscopic analysis (confocal microscopy) analysis has been shown to be several orders of magnitude more sensitive. In addition, because of its improved detection limits, the FDG substrate has found extensive use in automated ELISA type assay systems .
This kit uses the beta-galactoside analog fluorescein di-β-D-Galactopyranoside (FDG) in a protocol that sensitively distinguishes lacZ+ vs. lacZ- cells. See also Product M0250 for more information and references.
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- Cells should be washed 3X with sterile 1X PBS
- Add an appropriate volume of fixant (1.85% Formaldehyde/ 0.35% Glutaraldehyde)
- Incubate at room temperature for 30mins
- Remove fixant and wash cells 3X with 1X PBS
- Prepare 500uM Substrate reagent in PBS immediately prior to use and apply to cells
- Incubate at 37C for 30mins to 2 hours
- Analyze as required
Analysis should be carried out as soon as possible as fixing the cells will cause the dye to leak out of the cells.
For staining of tissue sections we recommend cryostat sections that have not been fixed, as fixation will lead to very diffuse staining. Tissue should be soaked in a solution of the substrate reagent at a concentration of 0.5 to 2 mM for 30 minutes to 2 hours at 37C. Tissue is then washed prior to observation. Keeping the Tissue cold will help prevent translocation of the fluorescent signal.
It is not unusual to see some background staining with this method, since the substrate in this kit, FDG, is quite labile. The obvious problems are from hydrolysis of the substrate prior to staining. If the staining solution is already quite yellow prior to staining, it may be already hydrolyzed and not usable. There is always a slight tan color, especially for the concentrated stock in the kit, but a bright yellow color indicates decomposition.
Our QC prior to shipment requires a background fluorescence reading of
There is a slight chance that there is just endogenous b-Gal activity (usually lysosomal activity) that is responsible for the high background. We include in the kit the inhibitor, chloroquine, which is a specific inhibitor of this activity. You might try that protocol. Additional inhibitors for this type of assay include verapamil and probenicid. Please see this article for more information about these techniques.
The IPTG is an broad based inhibitor and is not really useful for live cell analysis. It is useful for kinetic assays to "stop" the enzyme completely.
- Pellet the yeast cells by centrifugation.
- Remove growth medium without disturbing pellet.
- Resuspend pellet in 1 ml of 70% ice cold ethanol.
- Shake vigorously for 5 minutes on ice.
- Centrifuge the cells again.
- Remove 70% ethanol without disturbing cell pellet.
- Resuspend the pellet in ice-cold 100 ul Z buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgSO4, and 0.27% mercaptoethanol. The mercaptoethanol should be added right before use. Z buffer without mercaptoethanol should be stored at 4C).
- Immediately add 100 ul of ice-cold 100 uM FDG in 100 ul Z buffer. The FDG may be prepared by diluting 10 ul of the 10 mM stock FDG substrate provided with the kit into 990 ul of Z buffer.
- Incubate for 20-30 minutes at 37C.
- Measure fluorescence by flow cytometry or microscopy.
The reaction may be stopped via the addition of Na2CO3 (142 mM final concentration), otherwise the reactions will require careful timing in order to ensure all reactions are analyzed at the same time after addition of the FDG substrate.
Please note Z buffer and 70% ethanol are not included in this kit.
The kit should be fine, if left out for a few hours at room temperature. Certainly store it in the freezer (-20oC) upon receipt until use.
The only potentially temperature-sensitive sample in the kit is the fluorescent substrate reagent. Evidence of decomposition will be evident by a change in color of the fluorescent substrate reagent solution to a bright yellow (fluorescent) color and a noticeably high background level of fluorescence for the "blank" samples. Please note: this solution normally has a tan or light yellow color because it is so concentrated.