来自大肠杆菌的 β-半乳糖苷酶基因被广泛用作报告基因检测标记。尽管 X-gal 是众所周知的用于检测细胞或组织样品中的 β-半乳糖苷酶的试剂，但由于细胞渗透性差，使用这些试剂的测定需要固定细胞或组织。此外，迄今为止，使用荧光试剂开发的检测方法不能清楚地区分β-半乳糖苷酶表达的细胞或区域。为了克服这些问题，Urano、Kamiya 及其同事成功开发了 SPiDER-βGal。 SPiDER-βGal理想地具有细胞渗透性和保留在细胞内区域的能力。1）通过酶促反应，当蛋白质在分子附近含有亲核官能团时，SPiDER-βGal立即形成醌甲基化物，充当亲电子试剂。通过探针与蛋白质发生反应，结合物变成荧光化合物。因此，SPIDER-βGal 允许进行单细胞分析，因为它会自我固定到细胞内蛋白质上。
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 37oC.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 37oC.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 37oC .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.
1) T. Doura, M. Kamiya, F. Obata, Y. Yamaguchi, T. Y. Hiyama, T. Matsuda, A. Fukamizu, M. Noda, M. Miura, Y. Urano, “Detection of LacZ-Positive Cells in Living Tissue with Single-Cell Resolution.”, Angew Chem Int Ed Engl., 2016, doi: 10.1002/anie.2016033282) H. Omori, S. Ogaki, D. Sakano, M. Sato, K. Umeda, N. Takeda, N. Nakagata, S. Kume, “Changes in expression of C2cd4c in pancreatic endocrine cells during pancreatic development.”, FEBS Lett., 2016, doi: 10.1002/1873-3468.122713) Y. Nakamura, A. Mochida, T. Nagaya, S. Okuyama, F. Ogata, P. L. Choyke, H. Kobayashi, “A topically-sprayable, activatable fluorescent and retaining probe,SPiDER-βGal for detecting cancer; Advantages of anchoring to cellular proteins after activation”, Oncotarget, 2017, doi: 10.18632/oncotarget.170804) S. Lu, S. Liu, A. Wietelmann, B. Kojonazarov, A. Atzberger, C. Tang, R. T. Schermuly, H. J. Grone, S. Offermanns, “Developmental vascular remodeling defects and postnatal kidney failure in mice lacking Gpr116 (Adgrf5) and Eltd1 (Adgrl4)”, PLoS ONE., 2017, 10.1371/journal.pone.0183166.5) 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/s41514-017-0012-0.6) Y. Nakatani, H. Kiyonari and T. Kondo , “Ecrg4 deficiency results in extended replicative capacity of neural stem cells in a Foxg1-dependent manner”, Development.,2019,doi: 10.1242/dev.168120 .7) A. A.Tokmakov AA and K. I. Sato , “Activity and intracellular localization of senescence-associated β-galactosidase in aging Xenopus oocytes and eggs.”, Exp. Gerontol.., 2019, 119, 157.8) Y. Han, T. Bedarida, Ye Ding, Q. Wang, P. Song, and M. H. Zou, “β-Hydroxybutyrate Prevents Vascular Senescence through hnRNP A1-Mediated Upregulation of Oct4”, Molecular Cell., 2019, 71, 1064–1078.
Mouse Tissue1) Live Staining・Dissect lacZ-expressed tissue from mouse , and cut it to the appropriate size.・Incubate in OPTI-MEM containing 10-20 μmol/l SPiDER-βGal at 37 degree in 5% CO
2. (It is possible to fix with 4% paraformaldehyde (PFA) after staining.)2) Fixed Tissue Staining・Dissect lacZ-expressed tissue from mouse , and cut it to the appropriate size.・Fix with a solution containing 1% PFA and 0.2% GA , and then incubate in PBS containing 10-20 μmol/l SPiDER-βGal for 60 minutes at room temperature.
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.
Is there comparison data with X-Gal? What is an advantage over X-Gal?
SPiDER-βGal allows stain of a sample with a shorter time and without fixing the sample.Please check the comparison data below. There is comparison data between SPiDER-βGal and X-Gal for tissue staining.【Mouse Kidney】Refer to Figure S14 and 15 on the Supporting Information.【Mouse Salivary Gland】Refer to Figure S14 and 15 on the Supporting Information.【Mouse Brain】Refer to Figure S16 and 17 on the Supporting Information.
What is an advantage over commercially available β-Galactosidase detection probe?
SPiDER-βGal has higher cell permeability and intracellular retention than any other commercially available probe, which allows stain of a sample with a shorter time and lower concentration for mammalian cells. Please check the Selection Guide for more details.
Can I use SPiDER-βGal for tissue staining?
Yes, SPiDER-βGal can be used for tissue staining. We have experimental data with the following samples:【Drosophila Tissue】Refer to Figure S9, 12 and 13 on the Supporting Information.【Mouse Kidney】Refer to Figure S14 and 15 on the Supporting Information.【Mouse Salivary Gland】Refer to Figure S14 and 15 on the Supporting Information.【Mouse Brain】Refer to Figure S16 on the Supporting Information.
Can I fix a sample after staining with SPiDER-βGal?
Yes, the sample stained with SPiDER-βGal can be fixed with 4% PFA or methanol.
Can I stain a fixed sample with SPiDER-βGal?
Yes, SPiDER-βGal can be used for the fixed sample. We have experimental data for HEK cell, drosophila and mouse tissue. However, since fixation causes lower β-galactosidase activity, please optimize the condition for fixation.
How many time can I assay with 1 kit (20 μg×3) ?
If the final concentration is prepared at 1 μmol/l for cell staining, you can do approximately fifty 35mm dishes, fifty 8-chamber plates and ten 96- well plates.
Is there information regarding cytotoxicity?
Cytotoxicity of SPiDER-βGal to HEK-lacZ(+) cells and HEK cells was measured with Cell Counting Kit-8.【Cytotoxicity assay with HEK-lacZ(+) cells and HEK cells】Refer to Figure S8 on the Supporting Information.
How long is the Working solution stable?
You can’t store the working solution. Please prepare the working solution prior to use.
What buffer can I use for preparing the Working solution？
You can use PBS, Hanks’ HEPES and HBSS etc.
What is the recommended filter?
Fluorescence microscopy: Ex.550/25 nm, Em.605/70 nmFlow Cytometry: Ex.488 nm, Em.530/30 nm
