Quantitative assays for beta-gal activity are carried out in 96 well plates using a commercial chemiluminescent assay system with prolonged (glow) emission. Although the FACS-based assay is important for characterization at the single cell level of live cells expressing beta-gal chimeric proteins, it is difficult to prepare more than about 150 samples at a time for FACS analysis. The chemiluminescent assay described here permits the analysis of thousands of samples in one experiment. Numerical results can be obtained one hour after the assay is begun.
Subconfluent tissue culture cells are trypsinized, counted in a Coulter Z1, and replated in white 96 well plates at 10,000 cells per well (for C2F3 mouse myoblasts) in a volume of 100 ml the day before the assay. Plates were maintained in appropriate media and incubator conditions (DME with 20% FBS at 37oC in 10% CO2 for C2F3 cells) up to the addition of the chemiluminescent reagent. We have tested white 96 well plates with white opaque bottoms and clear bottoms (which permit viewing cells on a microscope). Opaque plates yield higher absolute luminescence values, however, the signal to background ratio in the two types of plates are similar. In clear bottom plates, luminescence from a strongly luminescent sample can be measured in a adjoining empty or weakly luminescent well. Such crosstalk is prevented in the fully opaque plates.
Cells are treated with agents that stimulate protein interactions of chimeric beta-gal proteins as desired. For example, cells are treated for minutes or hours with a growth factor that induces dimerization of a chimeric growth factor receptor - beta-gal protein. Normally, addition of agents is accomplished by replacing the media in the well with fresh media containing the agent, maintaining a volume of 100 ml per well. The media in untreated wells is also replaced at the same time, as background luminescence (luminescence measured in wells containing only media and the chemiluminescence reagents) increases the longer the media (DME in our case) is left in the well. Wells are grouped into triplicate or quadruplicate samples for treatment. If wells are to be treated for different times, the start of treatment must vary so that all treatments in a given plate end at the same time, or the different timepoints must be on separate plates. In the latter case, controls must be present on each plate to control for any variations in the chemiluminescent assay.
At the end of the treatment time, the Tropix Gal-Screen chemiluminescent reagent is added to the plates. Tropix Gal-Screen (GSY200 or GSY1000) consists of two components. The Gal-Screen substrate (1,2-dioxetane compound) is diluted 1:25 immediately before use into Gal-Screen buffer B equilibrated to room temperature. 100 ml of Gal-Screen is added to each well without removing the media. The plate is then incubated at 26oC to 28oC for 45 minutes to one hour, and is then read on a plate reader, measuring each well for 1 second. All readings, especially if multiple plates are used, are made in the period of time in which the chemiluminescence has plateaued, which is typically in the 45 minute to 2 hour range.
The following plate readers offer comparable sensitivity: Tropix TR717, EG&G Wallac Berthold LB96V, Wallac/EG&G MicroBeta Plus (current version is MicroBeta Trilux). All of these machines have optional injectors. On the MicroBeta, addition of an injector to the machine reduces the sensitivity of the instrument. On the TR717 and LB96V, the presence of an injector does not affect the sensitivity of the instrument. The MicroBeta has the advantage of being able to count multiple plates unattended. However, it takes 5 to 10 minutes to count one plate whereas the TR717 and LB96V, which only hold one plate at a time, requires about 2 minutes to count a plate. The data is then analyzed in a Microsoft Excel spreadsheet. Replicate samples are averaged together, and the luminescence value of wells containing only media, or containing cells lacking the beta-gal constructs, are subtracted from the measured values.