Detection of β-gal activity by chemiluminescent imaging (CLI) using β-gal enzyme and cultured cells.

a) The chemical structure of Galacto-Light PlusTM substrate; b) Differential light emission from wells containing various enzyme + substrate mixtures. Row A: 10 µl PBS; Row B: β-galactosidase (1 U in 10 µl PBS (pH: 7.2-7.4)); 1: +20 µl PBS; 2: +1 µl Galacto-plus (diluted to 10 µl in PBS)+10 µl PBS; 3: +10 µl reaction buffer) +10 µl PBS, 4: +10 µl accelerator buffer +10 µl PBS; 5: +1 µl Galacto-plus +10 µl reaction buffer +10 µl accelerator buffer (1∶10∶10); 6: +2 µl Galacto-plus +8 µl reaction buffer +10 µl accelerator buffer (1∶4∶5) (Total volume: 30 µl per well). c) CLI signal intensity for mixtures in (b) d) Varying numbers of MCF7-WT (upper A&B) and MCF7-lacZ (lower C&D) breast cancer cells in wells (0, 1×103, 5×103 ,1×104, 5×104, 1×105, 5×105, 1×106 cells, respectively) imaged using a sensitive CCD camera (exposure time 2 s) following addition of Galacto-Light PlusTM mixture (comprising 10 µl substrate +10 µl accelerant + buffer with (rows A,B E, F) or without (rows C, D, G, H) added lysis buffer); e) Signal intensities for MCF7-lacZ (▪) and -WT (Δ) cells in (d), where open symbols indicate inclusion of lysis buffer. Exposure times ranged from 5 s to 120 s to ensure adequate SNR without overloading.