Cell migration and invasion assays were performed according to a previous study [18 (link)]. Corning Transwell polycarbonate membrane cell culture inserts with 8.0-μm pores were uncoated for migration assay, or coated with 50 μL of BD Matrigel Basement Membrane Matrix (BD Biosciences, USA) diluted 1:3 with FBS-free DMEM (fetal bovine serum free Dulbecco’s Modified Eagle Medium) for invasion assay. Cells were trypsinized and then resuspended in serum-free medium. 2 × 104 cells were seeded in the top chamber with serum-free medium and allowed to migrate toward serum-containing medium in the lower chamber for 24 h. Then cells were fixed in 4% paraformaldehyde and stained with 0.01% crystal violet (AS1086, ASPEN, China) for 10 min. After wiping the cells remaining in the upper chamber with cotton swabs, the migratory or invasive cells were imaged and counted using a Leica AM6000 microscope (Leica Microsystems, Germany). The numbers of migratory or invasive cells were calculated as the average numbers of five random fields per well under 100× magnification.
Am6000 microscope
Manufactured by Leica
Sourced in Germany
The Leica AM6000 is a high-performance microscope designed for advanced laboratory applications. It features a sturdy construction, high-quality optics, and a range of specialized accessories to support various research and analysis needs. The core function of the Leica AM6000 is to provide users with a reliable and versatile imaging platform for detailed observation and examination of samples.
Automatically generated - may contain errors
Lab products found in correlation
Matrigel, by BD (8 mentions)
Crystal violet, by Merck Group (7 mentions)
Upper transwell chamber, by Corning (7 mentions)
Crystal violet, by Beyotime (5 mentions)
Upper transwell chamber, by BD (3 mentions)
Giemsa stain kit, by Abcam (3 mentions)
Beyoclick edu cell proliferation kit, by Beyotime (2 mentions)
Transwell polycarbonate membrane cell culture inserts, by BD (1 mentions)
As1086, by Aspen (1 mentions)
Matrigel basement membrane matrix, by BD (1 mentions)
Biocoat matrigel invasion chamber, by BD (1 mentions)
8 μm transwell chamber, by Corning (1 mentions)
Crystal violet staining solution, by Beyotime (1 mentions)
Transwell plate, by Corning (1 mentions)
Ab15580, by Abcam (1 mentions)
Keyfluor488 edu kit, by Keygen Biotech (1 mentions)
Annexin 5 fluorescein isothiocyanate apoptosis detection kit, by BD (1 mentions)
Facsan flow cytometry, by BD (1 mentions)
Rnascope kit, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
35 protocols using am6000 microscope
1
Cell Migration and Invasion Assays
2
Colony Formation Assay with rhCTGF
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The transfected cells (5×102 cells/well) were seeded in 6-well plates and co-cultured with or without rhCTGF for 14 days. The cell medium was refreshed every 3 days. On day 14, the cells were fixed with 4% paraformaldehyde at room temperature for 10 min and then stained with 0.5% crystal violet (Beyotime Institute of Biotechnology) for 20 min at ambient temperature. Subsequently, the number of colonies (with >50 cells considered to be a colony) was counted manually under a Leica AM6000 microscope (Leica Microsystems GmbH).
3
Colony Formation Assay Protocol
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Cell with indicated treatment were trypsinized and resuspended in culture medium. Cells were seeded into a 6-well plate (1000 cells/well) and cultured for 14 days, and the culture medium was changed every 3 days during the period. After 14 days, cells were fixed with 4% paraformaldehyde at room temperature for 10 mins and stained with Giemsa reagent (Giemsa Stain Kit, Abcam ab150670) or 0.5% crystal violet (Beyotime, Shanghai, China) for 20 mins. Subsequently, the number of colonies was counted and the morphology of the colonies was photographed under Leica AM6000 microscope (Leica, Wetzlar, Germany).
4
Quantifying Cell Proliferation with EdU Assay
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EdU incorporation assay was performed using Click‐iT™ EdU Cell Proliferation Kit for Imaging, Alexa Fluor™ 555 (Thermo Fisher Scietufic). Prewarmed the 2X EdU solution was mixed with an equal volume of culture medium in a 96‐well plate, and the cells were incubated for 2 h. The medium was discarded and cells were washed twice with PBS, followed by the fixation with 100 μL of 3.7% formaldehyde for 15 min. After the removal of fixative solution, cells were washed twice with 100 μL of PBS with 3% BSA. Then 100 μL of 0.5% Triton® X‐100 in PBS was added to each well for 20‐min incubation. After the removal of the solution, 1 x Click‐iT® reaction cocktail was prepared based on the manufacturer's instruction and added to cells for 30 min. After washing, cells were counter‐stained by 500 nM DAPI in PBS and the images were captured under Leica AM6000 microscope (Leica).
5
Transwell Assay for Cell Migration and Invasion
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The migration and invasion ability of cells were determined using Transwell assay. Cells with different treatments were collected and resuspended in serum‐free medium. The transwell upper chamber (Corning) without Matrigel (BD Biosciences) was used for migration assay, and the transwell upper chamber coated with Matrigel was used for invasion assay. About 5 × 105 cells were inoculated into the upper chamber in serum‐free medium and 500 μL of 20% serum‐containing medium was added to the lower chamber. After 18 h, the cells were fixed with 4% paraformaldehyde at room temperature for 10 min and stained with 0.5% crystal violet (Sigma) for 20 min. Cells were photographed under Leica AM6000 microscope (Leica), and the number of migrating and invading cells were counted from 5 randomly selected fields in each samples.
6
Transwell-based Cell Migration and Invasion Assay
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The migration and invasion abilities of cells after indicated treatments were determined using BD BioCoat™ BD Matrigel™ Invasion Chamber (BD Biosciences, Franklin Lakes, NJ, USA) in accordance with the manufacturer’s instructions. The transwell upper chamber without Matrigel (BD Biosciences, 356234) was used for migration assay, while transwell upper chamber coated with Matrigel was used for invasion assay. 6 × 105 Cells were inoculated into transwell upper chamber in serum-free medium and 1000 μL of 10% serum-containing medium was added to the lower chamber. After 24 hours, cells on the transwell membrane were fixed with 4% paraformaldehyde at room temperature for 10 mins and stained with 0.5% crystal violet (Beyotime, Shanghai, China)) for 15 mins. Cells were photographed under Leica AM6000 microscope (Leica, Wetzlar, Germany) at 400x magnification. The cell image was analyzed with Image J software (Bethesda, MD, USA).
7
EdU Proliferation Assay Protocol
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We performed EdU staining assay using the BeyoClick™ EdU Cell Proliferation Kit (Beyotime, Beijing, China) and carried out the assay in the relevant experimental groups according to the instructions of the kit. The EdU staining signal in the nuclei was detected by Leica AM6000 microscope (Leica, Wetzlar, Germany) at 100X magnification.
8
Transwell Assay for Cell Migration and Invasion
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BLCA cells were trypsinized and re-suspended in serum-free medium. The Transwell upper chamber (Corning, NY, USA) with the 8 μm pore size was used as the physical barrier for the migration assay, while the Transwell upper chamber coated with Matrigel (BD Biosciences, MA, USA) to mimic the extracellular matrix during tissue invasion was used for the invasion assay [29 (link)]. The cells in the serum-free medium were inoculated in the upper chamber at a cell density of 5 × 105 cells/ml. The lower chamber was filled with 600 μl of complete medium. After 24 hours, the cells on the Transwell membrane were fixed and stained with 0.1% crystal violet for 30 min. After washing, the migrating or invading cells on the membrane were observed under a light microscope. For quantification, 5 random fields of each sample were counted at 100X magnification using Leica AM6000 microscope (Leica, Wetzlar, Germany).
9
Cell Invasion and Migration Assay
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Cell invasion and migration assays were performed using the 8-μm Transwell chamber (Corning, NY, USA) and placed into the 24-well plates. Transwell coated with Matrigel (0.5 mg/mL, BD Biosciences) was used for invasion assay. PCL5 or HepG2 cells were trypsinized and suspended in FBS-free DMEM. 5 × 105 cells were inoculated in the upper Transwell chamber and DMEM with 10% FBS was added in the lower chamber as the chemoattractant. After 24 hours, culture medium was discarded and the cells were fixed with 4% paraformaldehyde at room temperature for 10 mins and stained with 0.5% crystal violet (Sigma, Germany) for 20 mins. Cells were photographed under Leica AM6000 microscope (Leica, Wetzlar, Germany), and the number of invading cells was counted.
10
Transwell Invasion and Migration Assay
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The transwell upper chamber (Corning, NY, USA, #3401) was used for invasion assay. The upper chamber surface was coated with 50 mg/L Matrigel at 1:8 dilution in cold PBS, and then filled with the diluted polycarbonate film, while the transwell upper chamber without Matrigel was used for migration assay. 5 × 105 cells were inoculated into the transwell upper chamber in serum-free medium and 2 mL of 10% serum-containing medium was added to the lower chamber. After incubation (24 hours for migration assays; 48 hours for invasion assays), culture medium was discarded and the cells were fixed with 4% paraformaldehyde at room temperature for 10 mins and stained with 0.5% crystal violet (Sigma, Germany, #109218) for 20 mins. Cells were photographed under Leica AM6000 microscope ((Leica, Wetzlar, Germany). Cells were counted and evaluated in five random fields of view.
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