The changes in the mitochondrial membrane potential were determined by JC-1, a fluorescent dye with potential-dependent accumulation in mitochondrial membrane as a monomer (fluoresced green) or dimer (fluoresced red-orange) in living cells as previously described (Wu et al., 2013 (link)). Briefly, the cells were seeded at a density of 2 × 105 cells/well in 6-well plates and treated with different concentrations of AS (25, 50, and 100 μM), TAK-242 (1 μM), or same volume of serum-free DMEM (vehicle control) for 12 h, then treated with Aβ1-42 (50 μM) or medium for another 24 h at 37°C. The cells were washed with PBS twice and incubated with JC-1 (5 μM) for 20 min at 37°C in a CO2 incubator, then washed with PBS three times for 5 min each time at 37°C in the dark. Fluorescent images were visualized and recorded under a fluorescence microscope at ×100 magnifications (AMG EVOS, Thermo Fisher Scientific, Inc., Waltham, MA, United States). All experiments were performed three times in triplicate.
Evos amg
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Evos AMG is a compact, inverted fluorescence microscope designed for live-cell imaging and analysis. It features a fully automated stage and objective turret, enabling precise and repeatable positioning and focusing. The Evos AMG is equipped with LED illumination, providing consistent and stable illumination for fluorescence imaging.
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Lab products found in correlation
7 protocols using evos amg
1
Mitochondrial Membrane Potential Analysis
2
Apoptosis Assessment in hBMECs
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Morphological assessment of apoptotic cells was processed by Hoechst 33258 staining method as described earlier (Yang et al., 2013 (link)). Briefly, hBMECs were seeded in 6-well culture plates at a density of 1 × 105 cells/well and treated with various concentrations of AS (25, 50, and 100 μM), TAK-242 (1 μM), or same volume of serum-free DMEM (vehicle control) for 12 h, then treated with Aβ1-42 (50 μM) or medium for another 24 h at 37°C. After incubating with fixative solution for 10 min, the medium was removed and washed with PBS for 15 min. Then, the cells were treated with 500 μL staining solution of Hoechst 33258 (10 μg/ml) for 5 min, followed by washing with PBS for 15 min at a dark room for reducing the background. Finally, the nuclear morphological changes of apoptotic cells were observed under a fluorescent microscope at ×400 magnifications (AMG EVOS, Thermo Fisher Scientific, Inc., Waltham, MA, United States) and the percentage of apoptotic cells was calculated according to the ratio of apoptotic cells to total cells. All experiments were performed three times in triplicate.
3
Fruit Fly Ovary Dissection and Imaging
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Female fruit flies were dissected in cold PBS, and ovaries were washed two times with PBS. Figures were captured under stereo microscope equipped with fluorescence adapter SFA-LFS-GR (Nightsea, United States) and an inverted fluorescence microscope (AMG EVOS, ThermoFisher, United States).
4
Vascular Sprouting of 3D Spheroids
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Tridimensional spheroids were generated by the hanging drop method. Drops (20 μL) of mbMSC and bmMSC suspensions (1.5 × 105 cells/drop) were laid on the inner surface of a Petri dish lid. To prevent hanging drop dry, 7 mL of PBS were added to Petri dishes and incubated in normoxic condition overnight at 37 °C in 5% CO2 for cellular aggregation. Spheroids were gently applied to a Flat bottom 96-well plate (Greiner Bio One, cat. 655986) previously coated with MatrigelTM Growth Factor Reduced (GFR) basement membrane matrix (BD Biosciences, San Jose, CA, US) and cultured in EGM-2 medium (Lonza, cat. CC-3156). Sprouting was evaluated after 48 h and 7 days. Images were acquired by phase-contrast inverted microscope Evos AMG (Thermo Fisher Scientific) and evaluated by measuring the distances between the spheroid edge and the last invading cell of sprouts by Image-Pro Plus software. After image analysis, 3D spheroids were cultured for more than 7 days and then were processed for immunofluorescence.
5
Fractionation and Immunoblotting of Cellular Proteins
6
Angiogenic Potential of HUVEC with MSC Conditioned Media
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Human umbilical vein endothelial cells at concentration of 2 × 104 cells per well were plated in 96-well plate (Corning®, Corning, NY, USA), coated with MatrigelTM GFR basement membrane matrix (BD Biosciences), in the presence of mbMSC and bmMSC conditioned medium, cultured for 48 or 72 h. Endothelial Growth Medium (EGM-2—Lonza, Basel, BS CH) was used as control. After 20 h, images at 10× magnification were acquired by phase-contrast inverted microscope Evos AMG (Thermo Fisher Scientific). Angiogenic capacity of HUVEC was analyzed by quantifying the total length of tubes and the number of branches in each experimental condition using the ImageJ software with angiogenesis analyzer extension.
7
Fractionation and Immunoblotting of Cellular Proteins
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For western blot analyses, cytoplasmic and nuclear extracts of cells were prepared using NE-PER fractionation kit followed by SDS-PAGE and immunoblotting. For whole cell lysates, cells were lysed in RIPA buffer. For IPs, cytoplasmic and nuclear extracts were diluted with NP40 lysis buffer [25mM Tris HCl (pH 7.5), 150mM NaCl, 1μM ZnCl2, 1% igepal (NP40)], supplemented with protease inhibitors, and incubated overnight at 4°C with the indicated antibodies and either Protein A agarose (for antibodies raised in rabbit) or Protein G agarose (for antibodies raised in mouse). IP eluates were subjected to SDS-PAGE and immunoblotting following three washes in IP lysis buffer. For immunofluorescence, cells were fixed in 4% paraformaldehyde, washed with PBS and permeabilized with 0.2% Triton X-100 in PBS for 10 minutes. Cells were blocked in Odyssey blocking buffer (Li-Cor, 927-4000) for 1 hour at room temperature followed by overnight incubation at 4°C in primary antibody. After washing with PBS, cells were incubated for 1 hour at room temperature in the dark in secondary antibodies. At last, cells were counterstained with DAPI (Thermo-Fisher). Images were acquired using an EVOS AMG (Thermo-Fisher) fluorescent microscope and representative images are shown at the same exposure and magnification.
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