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 cells1. 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.

Live Imaging of Drosophila tissueLiving drosophila tissue was incubated with 10 µmol/l SPiDER-βGal and 16 µmol/l Hoechst 33342 for 20-30 min, observed with confocal microscope.Only β-galactosidase expressed cells was detected with SPiDER-βGal.

Data was kindly provided by Dr. Y. Urano, at University of Tokyo, Graduate School of Medicine.

Imaging of a Fixed Tissue of a DrosophilaFormalin fixed drosophila generative cells were stained by SPiDER-βGal and DAPI. The β-galactosidase, which is expressed at the nucleus of drosophila generative cells, could be obtained by SPiDER-βGal.

Data was kindly provided by Dr. T. Nakamura, at University of Kumamoto, Institute of Molecular Embryology and Genetics.

Imaging of a Pancreas of a MouseSPiDER-βGal and Salmon-Gal were evaluated in the detectability of β-galactosidase on a pancreas of a mouse. SPiDER-βGal was able to detect the difference of the expression point of β-galactosidase more clearly than Salmon-Gal.

Data was kindly provided by Dr. S. Kume, at Tokyo Institute of Technology, School of Bioscience and Biotechnology.

Cell Senescence Assay with SPiDER-βGal by fluorescent microscopy and flow cytometrySA β-Gal in WI-38 cells induced by hydrogen peroxide was detected with SPiDER-βGal by fluorescent microscopy. SA β-Gal expression correlates with expression of other cellular senescence marker γ-H2AX. The increased expression of SA β-Gal was measured by flow cytometry.

Detection of SA-β-gal in the Tissue SampleReference paper using Dojindo’s SPiDER-β-gal for the tissue sample of diabetic mouse model was published.<Condition Tissue Samples were Labelled>Tissue sample was sliced into thin pieces after rapid freezing. The sliced samples were incubated in 4% Paraformaldehyde at room temperature for 20 minutes. First the samples were washed in PBS. Then, 20 μmol/l SPiDER-βGal was added and was incubated for 1 hour at 37℃. The samples were washed in PBS and observed under microscope.For more detail, please refer to the publication:T. Sugizaki, S. Zhu, G. Guo, A. Matsumoto, J. Zhao, M. Endo, H. Horiguchi, J. Morinaga, Z. Tian, T. Kadomatsu, K. Miyata, H. Itoh & Y. Oike,“Treatment of diabetic mice with the SGLT2 inhibitor TA-1887 antagonizes diabetic cachexia and decreases mortality”, Nature Partner Journal：Aging and Mechanisms of Disease., doi:10.1038/s41514-017-0012-0.

Drosophila Tissue1) Live Staining・Dissect lacZ-expressed tissue from Drosophila larvae , and then incubate in medium containing 10-20 μmol/l SPiDER-βGal for 20-30 minutes at room temperature.・If you would like to staining nuclear, please add 16 μmol/l Hoechst 33342 together. It is possible to fix with 4% paraformaldehyde (PFA) after staining.2) Fixed Tissue Staining・Dissect lacZ-expressed tissue from Drosophila larvae , and then fix for 20 minutes in 4% PFA in PBS.・After washing in PBS, incubate in PBS containing 10-20 μmol/l SPiDER-βGal for 20-30 minutes at room temperature.3) Combination with Immunostaining・Dissect lacZ-expressed tissue from Drosophila larvae , and then fix for 20 minutes in 4% PFA in PBS.・Wash with PBS-T for 3 times, 5 minutes each.・Dissolve primary-antibody in PBS-T and incubate for 30 minutes.・Wash with PBS-T for 3 times, 5 minutes each.・Incubate in PBS containg 10 μmol/l SPiDER-βGal, 16 μmol/l Hoechst 33342 and secondary antibody for 30 minutes.・Wash with PBS for 3 times, 5 minutes each.