Eu5888

Manufactured by Leica

Sourced in Germany

The EU5888 is a compact and portable laboratory equipment designed for routine laboratory tasks. It features a sturdy construction and intuitive controls for reliable performance.

Automatically generated - may contain errors

Lab products found in correlation

Fluorescence conjugated secondary antibody, by Beyotime (8 mentions) 4 6 diamidino 2 phenylindole (dapi), by Keygen Biotech (6 mentions) Anti runx2, by Abcam (4 mentions) Fluorescence microscope, by Leica (4 mentions) 2 4 amidinophenyl 6 indolecarbamidine dihydrochloride dapi, by Beyotime (3 mentions) Col1a1, by Leica (3 mentions) Col1a1, by Abcam (3 mentions) Rhodamine conjugated phalloidin, by Thermo Fisher Scientific (3 mentions) Inverted fluorescence microscope, by Leica (3 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions) Anti runx2, by Cell Signaling Technology (3 mentions) Anti col1a1, by Abcam (2 mentions) Dylight 550 conjugate, by Boster Bio (2 mentions) Paraformaldehyde (pfa), by Merck Group (2 mentions) S 4800, by Hitachi (2 mentions) β catenin, by Cell Signaling Technology (2 mentions) Fluorescence conjugated secondary antibody, by Jackson ImmunoResearch (2 mentions) 4 6 diamidino 2 phenylindole (dapi), by Roche (2 mentions) Anti foxa2, by Cell Signaling Technology (1 mentions) P erk, by Leica (1 mentions)

22 protocols using eu5888

Lentiviral GFP Transduction of hBMSCs

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A lentiviral green fluorescent protein (GFP) vector was purchased from Cyagen Biosciences and transfections were performed according to the manufacturer's instructions. Briefly, hBMSCs were incubated with GFP lentiviral particles and 5 μg polybrene/mL in growth medium. After 18 hours, the infection medium was replaced with hBMSC growth medium. After 2 days, the transfection results were observed by fluorescence microscopy (EU5888; Leica Camera, Wetzlar, Germany).
GFP-hBMSC 2D and 3D culture in GelMA For 2D culture, 10% GelMA was spread over 48-well plates and crosslinked using a 365-nm UV lamp. GFP-hBMSCs were then seeded on the surface of GelMA and cultured in growth medium at 37°C in an atmosphere of 5% CO 2 . For 3D culture, the cultured hBMSCs were trypsinised and collected by centrifugation. The resulting pellets were introduced onto 10% GelMA pre-solution and 0.25% (w/v) LAP as a photoinitiator, and sterilised with a 0.22 μm polyethersulfone (PES) membrane syringe filter (Guangzhou Jet Bio-Filtration). Finally, the material was crosslinked using a 365 nm UV lamp and washed twice before culturing the hydrogels in hBMSC growth medium at 37°C in an atmosphere of 5% CO 2 .

Xue D., Bai J., Xu J., Hang K., Kuang Z., Zhou C., Li Y., & Wang Y. (2020). Glycyrrhizic acid promotes osteogenic differentiation of human bone mesenchymal stem cells by activating the Wnt/β-catenin signalling pathway.

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Immunofluorescence Analysis of hBMSCs

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A 12-well plate was used to place hBMSCs in induction medium, and a fluorescence microscope (EU5888; Leica Camera, Wetzlar, Germany) was used for the evaluation of RUNX2 and COL1A1. At room temperature, the cells were treated with 4% paraformaldehyde. After 15 min, hBMSCs were permeabilized for 5 min with 0.1% Triton X-100 in PBS and blocked in 2% bovine serum albumin for 30 min. Fixed cells were washed and incubated overnight with anti-RUNX2 (1:500; CST) and anti-COL1A1 (1:500; CST). Then the fluorescence-conjugated secondary antibody (Beyotime) was added to the cells for 60 min, and DAPI (Nanjing KeyGen Biotech, Nanjing, China) was used to stain the nuclei for 5 min. The images were captured by a fluorescence microscope (Leica Camera) and the fields were selected randomly.

Xu J., Fu L., Bai J., Zhong H., Kuang Z., Zhou C., Hu B., Ni L., Ying L., Chen E., Zhang W., Wu J., Xue D., Li W, & Pan Z. (2021). Low-dose IL-34 has no effect on osteoclastogenesis but promotes osteogenesis of hBMSCs partly via activation of the PI3K/AKT and ERK signaling pathways. Stem Cell Research & Therapy, 12, 268.

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Lentiviral GFP Transduction of hBMSCs

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A lentiviral green fluorescent protein (GFP) vector was purchased from Cyagen Biosciences and transfections were performed according to the manufacturer’s instructions. Briefly, hBMSCs were incubated with GFP lentiviral particles and 5 μg polybrene/mL in growth medium. After 18 h, the infection medium was replaced with hBMSC growth medium. After 2 days, the GFP activity were observed by fluorescence microscopy (EU5888; Leica Camera, Wetzlar, Germany).

Bai J., Xu J., Hang K., Kuang Z., Ying L., Zhou C., Ni L., Wang Y, & Xue D. (2021). Glycyrrhizic Acid Promotes Osteogenic Differentiation of Human Bone Marrow Stromal Cells by Activating the Wnt/β-Catenin Signaling Pathway. Frontiers in Pharmacology, 12, 607635.

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Immunofluorescence Staining of Osteogenic Markers

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Cells were cultured in 12-well plates with OIM. After induction of osteogenesis, cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilised in 0.05% Triton X-100 for 30 minutes and blocked in 2% bovine serum albumin (BSA) for 30 minutes. Fixed cells were washed and incubated overnight with anti-RUNX2 (1:500; Abcam) or anti-COL1A1 (1:500; Abcam) antibody. The cells were then incubated with fluorescence-conjugated secondary antibody (DyLight 550 Conjugate; Boster Biological Technology, Wuhan, China) for 2 hours and nuclei were stained with 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) (Beyotime) for 5 minutes. Samples were observed under a fluorescence microscope (EU5888; Leica, Wetzlar, Germany).

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Immunofluorescence Profiling of Osteogenic Markers

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Cells were cultured in a 12-well plate, and FOXA2, RUNX2, COL1A1, t-ERK and p-ERK were detected using a fluorescence microscope (EU5888; Leica, Wetzlar, Germany). Briefly, cells were fixed in 4% paraformaldehyde (Sigma) for 15 min at room temperature, permeabilized and blocked for 30 min in 0.05% Triton X-100 and 5% bovine serum albumin (BSA). Fixed cells were washed three times with PBS and incubated at 4 °C overnight with anti-FOXA2 (1:400; Cell Signalling Technology), RUNX2 (1:1600; Cell Signalling Technology), COL1A1 (1:500; Abcam, Shanghai, China), t-ERK (1:800; Cell Signalling Technology) or p-ERK (1:200; Cell Signalling Technology). Cells were incubated with a fluorescence-conjugated secondary antibody (Beyotime) at room temperature for 2 h and nuclei were stained with 4′,6-diamidino-2-phenylindole (KeyGen Biotech, Nanjing, China) for 4 min. Samples were then observed and photographed under a fluorescence microscope (Leica).

Ye C., Chen M., Chen E., Li W., Wang S., Ding Q., Wang C., Zhou C., Tang L., Hou W., Hang K., He R., Pan Z, & Zhang W. (2018). Knockdown of FOXA2 enhances the osteogenic differentiation of bone marrow-derived mesenchymal stem cells partly via activation of the ERK signalling pathway. Cell Death & Disease, 9(8), 836.

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Visualization of F-Actin Rings in Osteoclasts

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To visualize F-actin rings, BMMs were treated with 25 ng⋅mL^-1 M-CSF and 50 ng⋅mL^-1 RANKL for 4 days. An equal number of BMM-derived osteoclasts were seeded onto bovine bone slices and allowed to adhere overnight. Cells were then treated with different concentrations of stevioside (0, 50, 100, and 200 μM) for another 2 days. Next, cells were fixed with 4% PFA for 15 min, permeabilized for 5 min with 0.5% Triton X-100, and stained with rhodamine-conjugated phalloidin (1:200; Invitrogen Life Technologies, Carlsbad, CA, United States) diluted in 0.2% bovine serum albumin (BSA)–PBS for 1 h. Fluorescent images were captured with a fluorescence microscope (EU5888, Leica, Wetzlar, Germany) and analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, United States). Then, these bone slices were washed twice with PBS for the resorption pit assay. Cells that had adhered to the bone slices were removed by mechanical agitation. Bone slice images were captured using a scanning electron microscope (SEM; S-4800, Hitachi, Japan) and analyzed using ImageJ software.

Meng J., Zhou C., Hu B., Luo M., Yang Y., Wang Y., Wang W., Jiang G., Hong J., Li S., Wu H., Yan S, & Yan W. (2018). Stevioside Prevents Wear Particle-Induced Osteolysis by Inhibiting Osteoclastogenesis and Inflammatory Response via the Suppression of TAK1 Activation. Frontiers in Pharmacology, 9, 1053.

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Visualization of Autophagy and Wnt Signaling

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Cells were cultured in a 12-well plate. Microtubule-associated protein 1 light chain 3 (LC3; 14600-1-AP, ProteinTech, Wuhan, Hubei, China) and activated β-catenin were detected using a fluorescence microscope (EU5888; Leica). Briefly, after treatment, cells were fixed in 4% paraformaldehyde for 15 min. Next, they were blocked in 5% bovine serum albumin for 30 min and incubated overnight with anti-LC3 (1:400; Cell Signaling Technology, Shanghai, China) and activated β-catenin (1:100; Cell Signaling Technology) at 4°C. The next day, cells were washed with phosphate-buffered saline and incubated with a fluorescence-conjugated secondary antibody (Abcam, Shanghai, China) for 2–3 h at 37°C, and nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich, Shanghai, China) for 2 min. Cells were observed using an inverted fluorescence microscope (Leica).

Zhang W., Hou W., Chen M., Chen E., Xue D., Ye C., Li W, & Pan Z. (2020). Upregulation of Parkin Accelerates Osteoblastic Differentiation of Bone Marrow-Derived Mesenchymal Stem Cells and Bone Regeneration by Enhancing Autophagy and β-Catenin Signaling. Frontiers in Cell and Developmental Biology, 8, 576104.

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Quantitative Immunofluorescence Analysis of RUNX2, SIRT7, and β-Catenin in Cultured Cells

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Cells were cultured in induction medium in a 12-well plate and evaluated for RUNX2, SIRT7, and β-catenin using a fluorescence microscope (EU5888; Leica, Wetzlar, Germany) as follows. Cells were fixed in 4% paraformaldehyde for 15 min at room temperature, permeabilized, and blocked for 30 min in 0.05% Triton X-100 and 2% bovine serum albumin. Fixed cells were washed and incubated overnight with anti-RUNX2 (1 : 1600; Cell Signaling Technology), SIRT7 (10 μg/ml; Abcam), or non-phosphorylated (active) β-catenin (1 : 1600; Cell Signaling Technology). Cells were incubated with a fluorescence-conjugated secondary antibody (Beyotime) for 120 min, and nuclei were stained with 4′,6-diamidino-2-phenylindole (KeyGen Biotech, Nanjing, China) for 5 min. Samples were observed under a fluorescence microscope (Leica).

Chen E.E., Zhang W., Ye C.C., Gao X., Jiang L.L., Zhao T.T., Pan Z.Z, & Xue D.D. (2017). Knockdown of SIRT7 enhances the osteogenic differentiation of human bone marrow mesenchymal stem cells partly via activation of the Wnt/β-catenin signaling pathway. Cell Death & Disease, 8(9), e3042-.

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Immunofluorescence Analysis of RUNX2 and SIRT1

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Cells (3 × 10⁴/cm²) were cultured in induction medium in a 12-well plate, and runt-related transcription factor 2 (RUNX2) and SIRT1 were detected using a fluorescence microscope (EU5888; Leica) after 3 days’ induction. Briefly, the cells were fixed in 4% paraformaldehyde for 15 min at room temperature after treatment. They were then blocked for 30 min in 0.01% Triton X-100 and 5% bovine serum albumin. Fixed cells were washed and incubated overnight with anti-RUNX2 (#12556S; 1:400; Cell Signaling Technology, Shanghai, China) and SIRT1 (#8469S; 1:100; Cell Signaling Technology). Cells were then incubated with a fluorescence-conjugated secondary antibody (ab150077 or ab150075, Abcam, Shanghai, China) for 120 min at room temperature, and nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich, Shanghai, China) for 5 min; they were then observed using an inverted fluorescence microscope (Leica).

Hou W., Ye C., Chen M., Li W., Gao X., He R., Zheng Q, & Zhang W. (2019). Bergenin Activates SIRT1 as a Novel Therapeutic Agent for Osteogenesis of Bone Mesenchymal Stem Cells. Frontiers in Pharmacology, 10, 618.

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Osteogenic Differentiation Marker Analysis

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Cells were cultured in induction medium in a 12-well plate, and RUNX2, COL1A1, and β-catenin were detected using a fluorescence microscope (EU5888; Leica, Wetzlar, Germany). Briefly, cells were fixed in 4% paraformaldehyde for 15 min at room temperature, permeabilized, and blocked for 30 min in 0.05% Triton X-100 and 2% bovine serum albumin. Fixed cells were washed and incubated overnight with anti-RUNX2 (1:1600; Cell Signaling Technology, Shanghai, China), COL1A1 (1:100; Santa Cruz Biotechnology, Shanghai, China), or β-catenin (1:100; Cell Signaling Technology). Cells were incubated with a fluorescence-conjugated secondary antibody (Beyotime) for 120 min, and nuclei were stained with 4′,6-diamidino-2-phenylindole (KeyGen Biotech, Nanjing, China) for 2 min. Samples were observed under a fluorescence microscope (Leica).

Zhang W., Xue D., Yin H., Wang S., Li C., Chen E., Hu D., Tao Y., Yu J., Zheng Q., Gao X, & Pan Z. (2016). Overexpression of HSPA1A enhances the osteogenic differentiation of bone marrow mesenchymal stem cells via activation of the Wnt/β-catenin signaling pathway. Scientific Reports, 6, 27622.

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