Peripheral blood mononuclear cells from healthy donors were isolated by density gradient centrifugation (Lymphoprep, Axis-Shield PoC, Oslo, Norway), washed with PBS (GIBCO, Invitrogen, Carlsbad, CA) plus 20% FBS, and suspended in endothelial cell basal medium-2 (EBM-2) (Lonza, Allendale, NJ, USA) supplemented with single aliquots of endothelial cell growth medium (EGM-2) (VEGF, human EGF-B, recombinant IGF-1, human EGF, heparin, ascorbic acid, and GA-100, Lonza) and 15% autologous plasma. Mononuclear cells were plated onto fibronectin (Biocoat, BD Biosciences, Franklin Lakes, NJ, USA) in coated, six-well plates at a density of 5 × 106 cells/well. We then incubated the fibronectin-coated plates at 37°C in a 5% CO2 atmosphere; 4 days later, we removed the cells in suspension, and the fraction of attached cells was cultivated with EBM-2 supplemented with 15% autologous plasma. The medium was recharged every 2 days for 3–4 weeks. After this period, EPCs could be visualized with an optical microscope (OPTIKA Microscopes, Italy) in the form of colonies (colony forming units, CFUs). The EPC phenotype (CD34+CD133+VEGFR2+) was verified with a cellular purity of >90%.
Optical microscope
Manufactured by Optika
Sourced in Italy
The optical microscope is a laboratory instrument used to magnify and observe small objects or details that are not visible to the naked eye. It utilizes lenses to focus light and produce a magnified image of the specimen under investigation.
Automatically generated - may contain errors
Lab products found in correlation
Human egf, by Lonza (1 mentions)
Ga 100, by Lonza (1 mentions)
Heparin, by Lonza (1 mentions)
Lymphoprep, by Axis-Shield (1 mentions)
Ebm 2, by Lonza (1 mentions)
Vascular endothelial growth factor (vegf), by Lonza (1 mentions)
Ascorbic acid, by Lonza (1 mentions)
Vision lite software, by Optika (1 mentions)
Jsm 7600f, by JEOL (1 mentions)
Pro view software, by Optika (1 mentions)
Rabbit anti mpo, by Abcam (1 mentions)
10 protocols using optical microscope
1
Isolation and Culture of Endothelial Progenitor Cells
2
Identification of Fungal Isolates by Morphology
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The identification of isolates was based on morphological characteristics, growth speed, colony and reverse colony colour, and colony texture, dimensions, and pigmentation. The identification of isolates was performed according to the method of [38 (link)]. The isolates were stained with lactophenol (Merck, Darmstadt, Germany) and observed under an optical microscope (Optika, Ponteranica, Italy). For the absence or presence of partitions, the colour of the mycelial filaments, the mode of the ramification of the septums, and the differentiation of spores were analysed as previously described [42 ].
3
Microgel Structural Characterization
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Microgels recovered in collecting baths were observed using an optical microscope (Optika, Italy). The size of microgels (N = 30) was determined using Optika Vision Lite software.
Scanning electron microscopy SEM observations on freeze-dried microgels were performed using JEOL JSM 7600F instrument (JEOL, Japan) working at an accelerating voltage of 5KV.
Images were collected by the Everhart-Thornley secondary electron detector of the chamber, far enough of the lens to ensure good depth of field.
Scanning electron microscopy SEM observations on freeze-dried microgels were performed using JEOL JSM 7600F instrument (JEOL, Japan) working at an accelerating voltage of 5KV.
Images were collected by the Everhart-Thornley secondary electron detector of the chamber, far enough of the lens to ensure good depth of field.
4
Colony Formation Assay Protocol
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For the colony formation assay, cells were seeded in triplicate in 60 mm dishes (5 × 102 cells/dishes). Cells were incubated for 8 days at 37 °C and stained with violet crystal solution (0.05% violet crystal w/v, 1% formaldehyde, 1% PBS, 1% methanol, and deionized water) for 20 min at room temperature. The number of colonies was manually quantified. Demonstrative images of the colonies were obtained under an optical microscope (Optika Italy) and using Optika Proview software.
5
Immunohistochemical Analysis of PLIN-2 and MPO
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A small portion of hepatic tissue was paraffin-embedded for the immunohistochemical (IHC) analysis of PLIN-2 and myeloperoxidase proteins. Then, 5 μm slices were cut with a microtome MR 2258 (Histo-Line Laboratories, Pantigliate, Milan, Italy). Antigen retrieval was performed in sodium citrate buffer pH 6 at 63 °C. Next, tissue slices were permeabilized with 0.2% Triton X100 in PBS. A total of 5% FBS was used as saturation buffer for 30 min. The following primary antibodies were used: rabbit anti-PLIN-2 (ABclonal, Woburn, MA, USA) and rabbit anti-MPO (Abcam, Cambridge, UK). The secondary HRP-conjugated polyclonal anti-rabbit IGg (KPL, Seracare, Milford, MA, USA) was used. To counterstain, nuclei hematoxylin was used. The IHC images were acquired by means of an optical microscope (Optika, Ponteranica, Bergamo, Italy) and analyzed by ImageJ software (version 1.52t, NIH, Bethesda, MD, USA).
6
Electrospun Fibrous Scaffold Fabrication
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Electrospinning was performed using a horizontal electrospinning setup (NanoNC, Seoul, Korea). The syringe tip was positioned approximately 10 cm above the flat metallic platform. A voltage of 15–20 kV was used to charge the solution. The solution was dispensed from a single nozzle spinneret of 25 gauzes (NanoNC, Seoul, Korea) at a constant feed rate of 0.2 mL/h at 22 °C and humidity of 12.5% ± 2.5%. Fiber morphologies in scaffolds were observed using an Optical microscope (Optical microscope, OPTIKA, Ponteranica, Italy), then fiber diameters and pore sizes were measured by an image software (ImageView Ver.3.7, Touptek Photonics, Hangzhou, China). The average values of fiber diameter and pore size were calculated by measuring five locations on the image and averaging them.
7
Optical Microscope Droplet Analysis
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Microscopic analysis was performed with an optical microscope (Optika, Ponteranica, Italy) equipped with 40x and 100x objectives and Optikam 3 digital camera. The images were analyzed using Image Tool®, version 3.0 software to determinate the size of the droplets.
8
Wound Healing Assay with HUVECs and HAECs
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HUVECs and HAECs were seeded into 6-well plates at a density of 4 × 105 cells per well. After 24 h of incubation, confluent cell monolayers were scratched across the midline using a pipette tip, and then scanned at 0, 6, 9, and 12 h using an optical microscope (Optika).
9
Electrospinning Deposition of PCL Fibers
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Electrospun fibers were deposited on a rod using an electrospinning device (NanoNC, Seoul, Korea) (Figure 2 ). The flow rate of the electrospinning solvent was fixed at 0.5 mL/h, and the distance and angle between the spinning nozzle and the rod were 60 mm and 35°, respectively. The operating voltage was set to 9 kV, and the rotating speed of the rod was 10 rpm (revolutions per min). A mixture of chloroform and methanol (4:1) was used as the solvent to dissolve the PCL. The morphologies and sizes of the electrospun fibers were observed using an optical microscope (Optika, Bergamo, Italy).
10
Microfluidic Emulsion Generation Protocol
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Chips with 50 or 200 µm internal diameter channels and two inlet and one outlet (Figure S1A), and three inlet and one outlet points (Figure S1B) were used for generating the water-inoil emulsions (W/O). The composition of the continuous and dispersed phases used for the different assays is shown in Table 1. Both the continuous and dispersed phases were injected with glass syringes (1 mL and 100 µL, respectively) using Nemesys Apparatus pumps controlled through a Nemesys software (Nemesys Interface, Madrid, Spain). Capillary polyethylene tubes (internal diameter: 0.38 mm) were used to connect the syringes to the chips' inlet and outlet points. W/O emulsions were collected off-chip. The formation of the emulsions in the chips was monitored in real time in an optical microscope (Optika, Ponteranica, Italy) connected to a "pike" camera using Vimba software (Vimba v3.1 ARM64, Germany).
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