Adipose tissue was obtained from the liposuction operations performed in the First Affiliated Hospital of Wenzhou Medical University in the period from November 2020 to December. The patients were randomly selected and included five adult females aged from 23 to 45. All patients signed the informed consent for scientific research and publication. Isolation of ADSCs was performed as described in our previous work [25 ]. Briefly, adipose tissue was fully cut with a scissor and digested using 0.1% collagenase type I (SCR103, Sigma, USA) at 37°C for 40 min. Then, the mixture was sequentially filtered using a 100 and a 200 mesh strainers. The filtrate was collected and centrifuged at room temperature at 250 g for 5 min. Finally, the pellet was resuspended in complete medium (HUXMD-90,011, Cyagen Biosciences, USA) and transferred into a plastic cell culture flask. The cells were cultured at 37°C with 5% CO2, and observed under a CKX41 inverted phase-contrast microscope (Olympus, JPN). The medium was changed every 3 days, and cells were passaged when reaching 90% confluence. The 3rd passage ADSCs were used for cell identification and d-ECM preparation, and the 5th passage ones were used for the subsequent experiments.
Ckx41 inverted phase contrast microscope
Manufactured by Olympus
Sourced in Japan
The CKX41 inverted phase contrast microscope is a compact and versatile lab equipment designed for routine microscopy applications. It features phase contrast optics for enhanced contrast and visualization of unstained samples. The microscope offers a sturdy construction and a range of objective magnifications to facilitate various observation requirements.
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Lab products found in correlation
Huxmd 90011, by Cyagen (1 mentions)
Scr103, by Merck Group (1 mentions)
Matrigel, by BD (1 mentions)
Transwell insert, by Corning (1 mentions)
Spark 10m microplate reader, by Tecan (1 mentions)
Ht7700, by Hitachi (1 mentions)
Tm3030plus, by Hitachi (1 mentions)
Peg nhs, by Merck Group (1 mentions)
Ab150680, by Abcam (1 mentions)
7 protocols using ckx41 inverted phase contrast microscope
1
Isolation and Characterization of ADSCs
2
Transwell Assay for Cell Migration and Invasion
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The capacities for cell migration and invasion were analyzed by the transwell assay, which was carried out using transwell inserts (8 μm pore size; Corning Incorporated, Corning, NY, USA) according to the manufacturer’s protocol. For the migration assay, cells resuspended in serum-free medium were placed in the upper chamber without Martigel coating. For the invasion assay, cells were placed in the upper chamber, which was coated with Matrigel (BD Biosciences, San Jose, CA, USA). Then, the chambers were incubated in culture medium with 10% FBS at 37°C in the lower chamber. After 24 hours, cells that did not migrate or invade were wiped out while the migrated and invaded cells were stained with 0.05% crystal violet for 30 minutes. Pictures were captured from five random fields under Olympus CKX41 inverted/phase-contrast microscope (Olympus Corporation, Toyko, Japan). Each experiment was repeated at least three times.
3
Embryoid Body Formation via Hanging Drop
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Murine embryonic and induced stem cells were differentiated through the hanging drop technique [63 (link),65 (link),66 (link)]. Trypsinized stem cells were resuspended in medium that did not contain LIF [high glucose (25 mM) DMEM that was supplemented with 15% (v/v) embryonic stem cell FBS, 1.0 mM sodium pyruvate, 0.1 mM non-essential amino acids and 0.1 mM 2-mercaptoethanol]. Cells were counted and diluted to a concentration of 25,000 cells/mL (or 500 cells per 20 μL drop). Drops of 20 μL were plated onto the lid of a 90-mm bacterial culture plate. The lid was then placed on top of a plate that contained 10 mL of PBS. Hanging drops were incubated at 37 C in 5% CO2, with 95% humidity for a period of 3–4 days [63 (link)]. Embryoid bodies were then transferred into 24, 48 or 96 well cell culture plates (Greiner Bio-One, #662160, Kremsmünster, Austria) that were treated with 0.1% gelatin. The embryoid bodies were either infected with reovirus directly as the cells were being transferred into the multiwell plates or after 1 or 10 days of differentiation. Spontaneous differentiation was confirmed by the presence of beating cardiomyocytes in cultures. Beating cells were identified through the use of an Olympus CKX41 inverted phase contrast microscope.
4
MTT Assay for Cytotoxicity Evaluation
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Cytotoxicity was assessed using the well-established MTT assay [31 (link)] as a conjugate measure for cell proliferation, viability and metabolic activity. Briefly, 10% (v/v) of MTT Reagent (1 mg/ml MTT in PBS) was added the cells incubated at 37 °C for 2 h. The reaction product was dissolved in 10% (m/v) SDS with 50% (v/v) dimethyl formamide in distilled water. Quantification was performed by absorption measurement at 550 nm with 630 nm reference wavelength using the Spark 10 M microplate reader (Tecan). The cells were also monitored for morphological changes using the CKX41 inverted phase contrast microscope (Olympus).
5
Visualizing Microgel Morphologies via Microscopy
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The optical microscopic morphologies of the reverse micelles in the emulsion and the microgels were visualized using an optical microscope (Olympus CKX41 Inverted Phase Contrast Microscope, Tokyo, Japan) at the magnification of 20×. Morphologies of the freeze-dried microgels were studied by scanning electron microscopy (Desktop SEM Hitachi TM3030 Plus, Tokyo, Japan) and transmission electron microscopy (TEM Hitachi HT7700, Tokyo, Japan). The microgels were freeze-dried by immersing in liquid N2 for 1 h before immediately loading the frozen samples into a chamber of a freeze dryer (Eyela Freeze Dryer FDU-1200, Tokyo, Japan). The freeze-drying process was operated at a condenser temperature of −40 °C under high vacuum. For SEM, the freeze-dried microgels were coated with gold using a gold sputter (Quick cool coater SC-701MC, Tokyo, Japan) under a high-vacuum condition. The surface morphology of the coated microgels was then observed at a voltage of 15 kV using a back-scatter detector (BSE) mode at 2000×. For TEM, the freeze-dried microgels were stained with Osmium tetroxide for 20 s before observing the sample morphology at 100 kV and 4000×.
6
Spheroids Encapsulation in PEGylated Fibrin Hydrogels
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To evaluate the spheroids’ ability to form a capillary-like network, they were encapsulated within PEGylated fibrin hydrogels as previously described (Gorkun et al., 2018 (link)). PEGylated fibrin hydrogel was prepared according to the previously developed protocol (Shpichka et al., 2020 (link)). Briefly, fibrinogen was covalently bonded with polyethylene glycol (PEG) using O,O′-bis[2-(N-succinimidyl-succinylamino)ethyl]polyethylene glycol (PEG-NHS; Sigma-Aldrich, Germany) at a molar ratio of 5:1 (PEG-NHS: fibrinogen). The reaction mixture was incubated at 37°C for 2 h. The spheroids suspension was distributed in fibrinogen solution, and then the thrombin solution was added (fibrinogen to thrombin ratio 1:1). This mixture immediately formed a gel.
Spheroids from all four groups were cultured in gels for 7 days, in complete growth medium supplemented with 10 ng/ml of VEGF, changed every 2 days. The process of tubule growth was monitored using a CKX41 inverted phase-contrast microscope (Olympus, Japan).
Spheroids from all four groups were cultured in gels for 7 days, in complete growth medium supplemented with 10 ng/ml of VEGF, changed every 2 days. The process of tubule growth was monitored using a CKX41 inverted phase-contrast microscope (Olympus, Japan).
7
Tissue Slide Staining Techniques
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Head tissue slides were stained with H&E using a standard procedure. The slides were treated with hematoxylin buffer at room temperature, rinsed three times with distilled water, and immersed in a 1% eosin Y solution. A PAS stain kit was used to stain the slides with PAS (ab150680; Abcam, Cambridge, UK). A conventional method was used to stain slides with toluidine blue. The slides were treated with toluidine blue working solution for 3 min. The slides were then washed three times with distilled water before being mounted and examined under a CKX41 inverted phase contrast microscope (Olympus, Tokyo, Japan).
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