I want to measure LacZ expression from an adenoviral vector in epithelial cells. I don't need anything particularly quantitative since I only want a yes or no answer as far as expression is concerned. I looked at a number of protocols that require fixation and permeabilization. Are there any protocols that allow you to add one compound (X-gal or a derivative) to live cells and get an answer?
I am not sure, but I think X-gal / IPTG assay, which is generally used for bacterial blue/white screening of positive clones could be useful here. But as I said, I am not sure. May be you can standardize something with this and come with a new protocol!
If you'd be willing to sacrifice a small population of your cells, you should be able to get actual Miller-unit quantification of the amount of LacZ activity in your cells. The protocol is quite easy and quick. The original "Miller" protocol, adapted for your purposes, is as follows (it looks more intensive than it really is; you're basically lysing cells with SDS then reading absorbance values in a spec.):
1.) Record OD600 of cell suspension (ideally between 0.5 and 1.0).
2.) In 15 mL Falcon tubes, add: Tube 1 --> 900 µL of Z buffer
Tube 2 --> 500 µL of Z buffer
3.) From the cell suspension, add: Tube 1 --> 100 µL of suspension (total volume now 1 mL)
Tube 2 --> 500 µL of suspension (total volume now 1 mL)
BLANK = 900 µL Z buffer + 100 µL resuspension buffer
4.) To each tube (including blank), add 50 µL of 0.1% SDS and 50 µL of chloroform, vortex, and incubate at 30 ºC in warmroom for 5 min.
5.) Start the reaction by adding 200 µL of ONPG (4 mg/mL) to each tube (including blank) and shake for a few seconds.
6.) IMMEDIATELY start timing the reaction.
7.) Once yellow colour has developed, add 500 µL of 1 M Na2CO3 solution to each tube (including blank), and STOP the timer.
8.) Using 1 mL of the stopped reaction volume for each tube, read the i) OD420 and OD550 on a standard benchtop spectrophotometer
--> Do not disturb the organic phase at the bottom of the reaction tube.
9.) Using the reaction time (Step 7) and the volume of cells used (Step 3), calculate the Miller units for β-galactosidase activity based on the following equation:
Miller Units = 1000 × [OD420 – (1.75 × OD550)]/(Time × Volume × OD600)
OD420 and OD550 = from Step 8
--> OD420 measures absorbance caused by the ONPG cleavage, and to a lesser extent by cell debris.
--> OD550 measures the amount of light scattering caused by cell debris, so this is used as a correction factor to determine the true amount of β-galactosidase activity.
--> Time = total reaction time, in minutes (Step 7).
--> Volume = volume of cells initially added from the resuspension, in mL (Step 3).
--> OD600 = initial OD reading of the resuspension at induction endpoint (Step 1).
Z buffer, per 50 mL:
O.80g Na2HPO4.7H2O (0.06M)
0.28g NaH2PO4.H2O (0.04M)
0.5 mL 1M KCl (0.01M)
0.05 mL 1M MgSO4 (0.001M)
0.135 mL b -mercaptoethanol (BME) (0.05M)
bring to approximately 40 mL with H2O, dissolve all the salts
adjust the pH to 7.0
use a graduated cylinder to bring the buffer to 50 mL
store at 4 C.
For a visual yes/no staining protocol I don't think you can avoid fixation. Mainly because the process of staining takes time and some of the components e.g. DMF are toxic. If you don't fix, by the time you come back to look all your cells have died and detached. As far as I know there is not a more time saving protocol.
If the fixation time really is a problem, perhaps it would be worth getting hold of an Ad.GFP reporter where you could instantly look and continually monitor.
Thanks for the advice. Toxic really isn't an issue since I don't care about the cells other than to check if the vector is working. My vector does give me GFP expression in my targeted cells, but doesn't express my inserted gene despite sequencing telling me that both the insert and the promoter are the correct sequence. Wanted to make sure my LacZ control insert was integrated and expressing, but I need to finish some other tests (i.e. PCR to determine that my purified vector actually still has my gene) before I get into that. Thanks for all the advice.
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