Description

The gene of β-galactosidase from E. coli is widely used as a reporter gene assay marker. Although X-gal is well known reagent to detect β-galactosidase in cell or tissue samples, the assay using these reagents require to fix cells or tissues due to the poor cell-permeability. In addition, so far developed the assay using fluorescence reagents cannot clearly differentiate β-galactosidase-expressed cells or regions.
To overcome these issues, Urano, Kamiya and co-workers have successfully developed SPiDER-βGal. SPiDER-βGal ideally possesses cell-permeability and the ability to retain in intracellular region.¹⁾
By the enzymatic reaction, SPiDER-βGal immediately forms a quinone methide that acts as electrophile when proteins containing nucleophilic functional groups nearby the molecules. By the probe undergoes the reaction with a protein, the conjugates become fluorescent compounds. Thus, SPiDER-βGal allows a single-cell analysis because it does self-immobilizing to the intracellular proteins.

Difference between SPiDER-βGal and C12FDG

Usage Examples:
Fluorescence microscopic detection of β-galactosidase-expressed cells

1. HEK cells at 5 × 10⁵ cells/ml (500 μl) and HEK/LacZ cells at 5 × 10⁵ cells/ml (500 μl) were seeded in a 35 mm dish in DMEM (10% fetal bovine serum, 1% penicillin-streptmycin) and cultured overnight in a 5% CO₂ incubator at 37^oC.
2. The cells were washed with 2 ml of Hanks’ HEPES buffer twice.
3. SPiDER-βGal working solution (2 ml) was added to the culture dish. The cells were then incubated for 15 minutes at 37^oC.
4. After the supernatant was removed, the cells were washed Hanks’ HEPES buffer (2 ml) twice.
5. Hanks’ HEPES buffer (2 ml) were added and the cells observed under a fluorescence microscope. (Fig. 3A)
6. After the supernatant was removed, 4% paraformaldehyde (PFA) /PBS solution (2 ml) was added to the culture dish. The cells were then incubated for 15 minutes at room temperature.
7. After 4% PFA/PBS solution was removed, the cells were washed Hanks’ HEPES buffer (2 ml) twice.
8. Hanks’ HEPES buffer (2 ml) were added and the cells observed under a fluorescence microscope. (Fig. 3B)

Flow cytometric detection of β-galactosidase-expressed cells
1. HEK cells at 5 × 10⁵ cells/ml (500 μl) and HEK/LacZ cells at 5 × 10⁵ cells/ml (500 μl) were mixed in a microtube.
2. SPiDER-βGal DMSO stock solution (1 μl) was added to the tube. The cells were then incubated 15 minutes at 37^oC .
3. The cells were analyzed under a flow cytometer. (488 nm excitation, 530/30 nm bandpass filter)

β-galactosidase-expressed cells (HEK/LacZ cells) were clearly differentiate from HEK cells in flow cytometry data analysis.