Wound-healing and Transwell migration assays were employed to assess the migration capacity of rDPCs after αMEM or rDFSC-CM treatment. For the wound-healing assay, rDPCs (2 × 105 cells/well) were seeded in 24-well plates in αMEM with 10% FBS for 24 h to obtain a monolayer. After the cells reached 90% confluence, a 4-μm-wide wound was made with a sterile pipette tip on the monolayer. The culture was replenished with αMEM or rDFSC-CM with or without 0.5 mg/L LPS. The cells were incubated at 37 °C in an atmosphere with 5% CO2 for 24 h. The migrated cells were captured under an inverted light microscope (Carl Zeiss, Germany). The Transwell migration assay was performed using Transwell chambers and inserts with 8-μm pores (Corning, USA). Suspended cells (at a density of 2 × 105 cells/mL in 100 μL of FBS-free medium) were seeded into the upper chambers. The lower chambers contained αMEM or rDFSC-CM with or without 0.5 mg/L LPS. After 24 h of incubation at 37 °C and 5% CO2, the cells were fixed with 4% paraformaldehyde (Beyotime, China) for 15 min and stained with a 1% crystal violet solution (Beyotime, China) for 30 min. The nonmigrated cells on the upper membrane were removed using cotton swabs. The cells that had migrated through the membrane were captured and counted under an inverted light microscope (Carl Zeiss, Germany).
Inverted light microscope
Manufactured by Zeiss
Sourced in Germany, United States
The Inverted light microscope is a type of optical microscope designed with the objective lens and the illumination system positioned below the specimen. This configuration allows for the observation of live cells and other samples in an upright position, which can be beneficial for certain applications.
Automatically generated - may contain errors
Lab products found in correlation
Matrigel, by BD (15 mentions)
Toluidine blue, by Merck Group (10 mentions)
Matrigel, by Merck Group (6 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Crystal violet, by Merck Group (2 mentions)
Cell counting kit 8 assay kit, by Dojindo Laboratories (2 mentions)
Antibody dilution buffer, by Agilent Technologies (2 mentions)
Target antigen retrieval solution, by Agilent Technologies (2 mentions)
3 3 diaminobenzidine (dab), by Agilent Technologies (2 mentions)
Transwell chamber, by Corning (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Transwell insert, by Corning (2 mentions)
F12 medium, by Thermo Fisher Scientific (1 mentions)
Hepatocyte growth factor (hgf), by Thermo Fisher Scientific (1 mentions)
D hanks solution, by Thermo Fisher Scientific (1 mentions)
Matrigel coated, by Corning (1 mentions)
8 mm transwell inserts, by Corning (1 mentions)
Crystal violet solution, by Beyotime (1 mentions)
Paraformaldehyde (pfa), by Beyotime (1 mentions)
Matrigel chamber, by BD (1 mentions)
74 protocols using inverted light microscope
1
Assessing Dermal Papilla Cell Migration
2
Evaluating SCAP Migration with EVs
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Wound-healing and Transwell migration assays were employed to assess the migration of SCAPs after EVs treatment. For wound-healing assay, SCAPs (2 × 105 cells/well) were cultured in 6-well plates for 24 h. When cells reached 90% confluence, a wound scratch was created with a sterile pipette tip, and culture medium was converted into FBS-free αMEM with or without EVs (20 µg/mL). Wound scratches were captured by inverted light microscope (Carl Zeiss, Oberkochen, Germany) after 24 h and the width of scratch was measured by Image J software (NIH, Bethesda, MD, USA).
In transwell migration assay, suspended cells (at a density of 2 × 105 cells/mL in 100 μL of FBS-free medium) were seeded into upper chamber of 8 μm pores Transwell inserts (Corning, NY, USA), while αMEM with or without EVs (20 µg/mL) were contained in lower chambers. Cells on top chamber were fixed with 4% paraformaldehyde after 24 h and stained with 0.1% crystal violet solution. The cells left on top membrane were gently removed with cotton swabs, and those migrated cells were captured and counted under an inverted light microscope (Carl Zeiss, Oberkochen, Germany).
In transwell migration assay, suspended cells (at a density of 2 × 105 cells/mL in 100 μL of FBS-free medium) were seeded into upper chamber of 8 μm pores Transwell inserts (Corning, NY, USA), while αMEM with or without EVs (20 µg/mL) were contained in lower chambers. Cells on top chamber were fixed with 4% paraformaldehyde after 24 h and stained with 0.1% crystal violet solution. The cells left on top membrane were gently removed with cotton swabs, and those migrated cells were captured and counted under an inverted light microscope (Carl Zeiss, Oberkochen, Germany).
3
Transwell Assay for Cell Migration
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After transfection for 24 h, PC cells were trypsinized and washed with D-Hanks solution (Gibco; Thermo Fisher Scientific, Inc.). Wells of 24-well plates were divided into an upper and bottom chamber with Matrigel inserts (pore size, 8 µm). The cell suspension of PC cells (5x105 cells) in DMEM was added to the upper chamber. F-12 medium (400 µl) supplemented with FBS (10%; Thermo Fisher Scientific, Inc.) and hepatocyte growth factor (20 ng/ml; Thermo Fisher Scientific, Inc.) was added to the bottom compartment. After incubation for 24 h at 37˚C, cells that migrated from the upper chamber to the lower one were stained with crystal violet. Cell migration was evaluated with a light inverted microscope (Zeiss AG; magnification, x200).
4
Cellular Interaction with Silver Nanoparticles
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Photographs of the interaction of living cells with the silver nanoparticles dispersions of different concentrations were taken over 72 h. The morphological changes were observed using a Carl Zeiss Jena (Germany) light inverted microscope at 70 × magnification.
5
Microplankton Enumeration and Pseudo-nitzschia Identification
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On arrival at the laboratory, the microplankton in a 50 mL subsample were allowed to settle on the base plate of a sedimentation chamber for a minimum of 20 h before analysis, following the method described by Utermöhl [50 (link)]. A Carl Zeiss inverted light microscope was used to identify and enumerate the microplankton in the subsample and cell counts were converted to numbers of cells per litre. During counting, the Pseudo-nitzschia were split into two groups based on valve width, as light microscopy does not allow identification to species level. Cells with a transapical axis of 3 µm or less and a needle-like appearance were considered to belong to the P. delicatissima complex, while those with a transapical axis greater than 3 µm and with convex valve margins were considered to belong to the P. seriata complex [29 (link)].
6
Transwell Invasion Assay Procedure
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The apical chamber surface of the bottom membrane of the Transwell chamber was coated with Matrigel (BD Biosciences) which was polymerized into a gel at 37°C for 30 minutes, with hydration of the basal membrane before use. The cells were cultured in serum‐free medium for 12 hours, followed by collection and resuspension of cells in serum‐free medium (1 × 105/mL). The lower chamber was added with 10% FBS, and 100 μL cell suspension was added into the Transwell chamber at 37°C for 24 hours. The cells that had not invaded the Matrigel membrane surface were gently removed with a cotton swab, and the remaining cells were fixed with 100% methanol and stained with 1% toluidine blue (Sigma‐Aldrich Chemical Company). Stained invading cells in five randomly selected areas were manually counted under an inverted light microscope (Carl Zeiss).
7
Tube Formation Assay for Angiogenesis
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Tube formation assay was performed as previously reported46 (link). Briefly, a total of 35 × 104 HUVECs were seeded in Matrigel-coated (Corning, USA) 96-well plates in DMEM-F12 including 1% EV-free FBS. The cells were treated with either 0.5 µg EVs derived from control or ticagrelor treated H9c2 cells. The tube formation was visualized at 6 h and 24 h of incubation by an inverted light microscope (Zeiss, Germany). Branche, mesh, segment, and node formations were analyzed by Angiogenesis Analyzer plug-in for ImageJ (NIH, USA)47 (link). Changes in angiogenesis on EV-treated cells were expressed as a percentage of the controls (untreated cells). Values represent the mean (± SD) of triplicate formation assay.
8
Transwell Assay for GBM Cell Migration and Invasion
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The migration and invasion ability of GBM cells was evaluated using Transwell assay by following the manufacturer’s protocol of 24-well plates and 8-mm transwell inserts (Corning Life Science). For migration analysis, GBM cells (5 × 104) suspending in 200 μL serum-free medium were seeded into the apical chambers and macrophages (1 × 104) were seeded into the basolateral chambers supplemented with 800 μL medium containing 10% FBS. For invasion analysis, the insert membranes were coated with Matrigel (50 mL/well, BD Bioscience, Franklin Lakes, NJ), which was allowed to polymerize at 37 °C for 30 min. The basement membrane was hydrated before use and the remaining steps were same as migration analysis. After culture for 24 h at 37 °C, non-migrating or non-invading cells were removed and then stained with 0.1% crystal violet for 30 min. Stained cells (migrated/invaded cells) were counted in five randomly selected fields under inverted light microscope (Carl Zeiss, German).
9
Transwell Invasion Assay Protocol
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Subsequent to 12‐h cell incubation in medium without serum, the harvested cells were resuspended in medium without serum (1 × 105 cells/mL). The medium encompassing 10% FBS was supplemented to the lower chamber. Cell suspension (100 µL) was supplemented to the Transwell chamber before 24‐h culture at 37ºC. Cells that did not invade the surface of the Matrigel membrane was gently removed with a cotton swab. The remaining cells were fixed with 100% methanol before 1% crystal violet (Sigma) staining. The stained cells were counted under an inverted light microscope (Carl Zeiss, Jena, Germany) in random 5 fields. Each experiment was repeated 3 times.
10
Endothelial Cell Viability and Angiogenesis
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After the cells were cultured on the sample surface for 1, 3, and 5 days, the viability of ECs was analyzed as described in “Cytotoxicity Analysis of OBs”. After culturing on the sample surface for 3 and 7 days, the enzyme-linked immunosorbent assay (ELISA) (R&D Systems, Minneapolis, MN, United States) was performed to measure the level of vascular endothelial growth factor (VEGF) secreted by ECs according to the manufacturer’s protocols, by which the results were normalized to the number of cells. The capillary tube formation of ECs was assessed by in vitro ECMatrixTM gel kit (Millipore, Billerica, MA, United States). An inverted light microscope (Zeiss) was used to capture the images of capillary-like networks from six randomly selected fields. Image J 1.45 software was applied to quantify the number of nodes, branches, and tubes.
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