The bioactivity of the EVs, the cECMH, and the PEG–cECMH with the encapsulated EVs (EVs–PEG–cECMH) was tested by investigating their anti-senescent effect in EDCs coming from two patients different from the EVs used as treatment. In triplicate, 250 µL samples of EVs (at 0.56 mg/mL) in SFM, cECM alone, or EVs in PEG–cECM were incubated for gelation during 24 h at 37 °C in 12-well Transwells® (3460, Corning®). EDCs from passage 3 were seeded at a concentration of 15,000 cells/cm2 in fibronectin precoated 12-well plates. The cells were left for 24 h to attach, and then the medium was changed to SFM (2 mL/well), and the Transwells® with SFM (controls), EVs in SFM, cECMH, or EVs–PEG–cECMH were added. After 72 h, the Transwells® were removed and the cells fixed and stained with the senescence-associated beta-galactosidase assay, following the manufacturer’s instructions (ab65351 Senescence Detection Kit, abcam®). A total of 14 images at 20x (around 650 cells) per well were taken with a Leica DMI3000B optical microscope and Leica DFC310 FX camera (Wetzlar, Germany), and analyzed using ImageJ Software. Cells were classified as senescent or non-senescent depending on whether they presented a blue color, and the percentage of senescent cells was calculated.
Dmi 3000b optical microscope
Manufactured by Leica
Sourced in Germany
The Leica DMI 3000 B is an optical microscope designed for routine applications. It features a compact, ergonomic design and offers basic illumination and observation options for a range of sample types.
Automatically generated - may contain errors
3 protocols using dmi 3000b optical microscope
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Anti-senescent Effects of EVs and Hydrogels
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Fluorescent Microscopy Protocols for Biomolecule Imaging
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Optical microscopy experiments were carried out on a Leica DMI 3000B optical microscope. Fluorescence imaging was performed using a Leica DFC 310FX set up, and dye molecules were excited by using specific filters with the following excitation (λex) and emission wavelength cut offs (λem); NBD-PE, λex = 463 nm, λem = 536 nm; FITC, λex = 492 nm and λem = 518 nm; TR-DHPE, λex = 582 nm and λem = 601 nm; rhodamine-B, λex = 528 nm and λem = 551 nm; RITC, λex = 570 nm and λem = 590 nm; resorufin, λex = 560 nm and λem = 572 nm; propidium iodide (PI), λex = 536 nm, λem = emission 617 nm. All glass slides used for the imaging were coated with BSA.
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Multimodal Optical Microscopy Techniques
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Optical microscopy experiments were carried out using a Leica DMI 3000B optical microscope. Fluorescence imaging was performed using a Leica DFC 310FX, and dye molecules were excited by using specific filters. Confocal microscopy imaging was performed on a Leica SP5-II laser scanning microscope attached to a Leica DMI 6000 inverted epifluorescence microscope and equipped with a X10 or X20 objective.
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