Migration of EPCs was evaluated by transwell migration assay (BD, San Diego, CA, USA). EPCs were treated as aforementioned. EPCs suspension (200 μL, 1.2 × 104 cells/mL in M199 medium) were added to the upper chamber of a 24-well transwell plate with 8 μm pore membrane. M199 and EGM-2MV medium were added to the upper and lower transwell chamber, respectively. After culture at 37 °C with 5% CO2 for 24 h, a cotton wool swab was used to gently wipe the upper cells that had not migrated, and the lower cells were fixed and stained with DAPI (Sigma, catalog number: D8417). The migratory EPCs in five randomly selected fields of view were analyzed under a fluorescence microscope (×100) (Eclipse TE300, Nikon, Tokyo, Japan).
Transwell migration assay
Manufactured by BD
Sourced in United States
The Transwell migration assay is a laboratory equipment used to measure the migratory capacity of cells. It consists of a two-chamber system separated by a porous membrane, allowing the study of cell migration in response to various stimuli.
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Lab products found in correlation
Eclipse te300, by Nikon (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Dm il led, by Leica (1 mentions)
Methocult, by STEMCELL (1 mentions)
Celltiter glo, by Promega (1 mentions)
Brdu cell proliferation assay, by Cell Signaling Technology (1 mentions)
Matrigel invasion assay, by BD (1 mentions)
Stereomicroscope, by Leica (1 mentions)
Crystal violet staining solution, by Beyotime (1 mentions)
8 μm pores, by BD (1 mentions)
10 protocols using transwell migration assay
1
Evaluating EPC Migration Dynamics
2
Transwell Migration Assay for PTS-Induced Invasiveness
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The effects of PTS on the invasive motility of Tca-8113 cells were determined using a transwell migration assay (BD Biosciences). In brief, after treatment with PTS for 1 h, cells (2.5×104) were seeded into the top chambers of a 24-well, 8-μm pore-size micropore polycarbonate membrane filter (BD Biosciences), and the bottom chambers were filled with 500 μl Dulbecco’s modified Eagle medium containing 10% FBS as a chemoattractant. Cells were allowed to migrate for 24 h at 37°C. Nonmigrated cells were removed with a cotton swab, and migrated cells were fixed with 4% paraformaldehyde and stained with 1% crystal violet. Data are represented as the average number of migrated cells per field (20 random ×20 magnification fields) per membrane filter.
3
Comprehensive Cell Characterization Assays
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Cell viability was analyzed by Cell Titer-Glo (CTG; Promega), and S-phase DNA synthesis by BrdU Cell Proliferation assay (Cell Signaling Technology). For colony-forming assay, 200 cells were plated in triplicate in 1 ml of mixture composed of 1.1% methylcellulose (MethoCultTM STEMCELL Technologies) in RPMI-1640 + 10% FBS. Crystal violet-stained colonies were scored after 2 weeks under an inverted microscope (Leica DM IL LED) at ×5 magnification. Cell migration was analyzed by Transwell migration assay (BD Biosciences) as described [16 (link)].
4
Transwell Migration Assay for Exosome-Induced Cell Movement
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Migration was determined using 8μ-pore filters for the Transwell migration assay (BD Biosciences), according to the manufacturer's instructions and as previously described [63 (link)]. Briefly, murine Raw264.7 cells and human pre-osteoclast cells (1×105 cells/well) treated for 24hrs with MM cell-derived exosomes (25μg/ml) were washed and resuspended in DMEM high glucose medium supplemented with 1% of ultracentifuged FBS. Cells were then placed in the upper migration chambers, whereas the lower chamber contained DMEM high glucose medium supplemented with 10% ultracentifugated FBS. Filters were removed after 24 hours, fixed in methanol and cells stained with Diff-Quick (Medion Diagnostics GmbH, Düdingen, Switzerland). Alternatively, pre-osteoclast cells were not treated and MM cell-derived exosomes (25μg/ml) were added directly in the lower chamber contained DMEM high glucose medium supplemented with 10% ultracentifugated FBS. Two independent experiments were performed in triplicate; cells from 5 different fields were counted for each condition.
5
Radiation-Induced Migration Assay
6
Transwell Chemotaxis Assay for miR-199a-5p
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Chemotaxis was determined using 8-u-pore filters for the Transwell migration assay (BD Biosciences), according to the manufacturer's instructions.
Briefly, OPM2 cells (1×105/well) transfected with either scrambled (miR-NC) or synthetic pre-miR-199a-5p (miR-199a-5p) were washed and resuspended in RPMI 1640 medium supplemented with 1% FBS. Then cells were placed in the upper migration chambers, whereas the lower chamber contained RPMI 1640 medium supplemented with 10% FBS. After 5 hours of incubation at 37°C-5%CO2, cells migrated to the lower chambers were determined by a Trypan-Blue exclusion assay. Huvec (7×103 cells/well) were resuspended in serum free RPMI 1640 medium supplemented with 0.1% BSA in transwell chemotaxis (BD Biosciences) above 8 um pore filters and exposed to conditioned medium from multiple myeloma cells transfected with either scrambled (miR-NC) or synthetic pre-miR-199a-5p (miR-199a-5p).
Filters were removed after 6 hours, fixed in methanol and stained with Diff-Quick (Medion Diagnostics GmbH). Two independent experiments were carried out in triplicate; cells from five different fields were counted for each condition.
Briefly, OPM2 cells (1×105/well) transfected with either scrambled (miR-NC) or synthetic pre-miR-199a-5p (miR-199a-5p) were washed and resuspended in RPMI 1640 medium supplemented with 1% FBS. Then cells were placed in the upper migration chambers, whereas the lower chamber contained RPMI 1640 medium supplemented with 10% FBS. After 5 hours of incubation at 37°C-5%CO2, cells migrated to the lower chambers were determined by a Trypan-Blue exclusion assay. Huvec (7×103 cells/well) were resuspended in serum free RPMI 1640 medium supplemented with 0.1% BSA in transwell chemotaxis (BD Biosciences) above 8 um pore filters and exposed to conditioned medium from multiple myeloma cells transfected with either scrambled (miR-NC) or synthetic pre-miR-199a-5p (miR-199a-5p).
Filters were removed after 6 hours, fixed in methanol and stained with Diff-Quick (Medion Diagnostics GmbH). Two independent experiments were carried out in triplicate; cells from five different fields were counted for each condition.
7
Transwell Cell Migration and Invasion Assay
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Cell migratory and invasive abilities were assessed by the transwell migration assay and Matrigel invasion assay (BD Biosciences, Franklin Lakes, NJ), respectively. Cells were trypsinized and were resuspended in serum-free DMEM. 1 × 105 cells in 100 μL of serum-free medium were seeded in the upper chamber of transwell chambers (8-μm pore membranes; Corning). Membranes were coated with 0.78 mg/ml Matrigel for invasion assay. After 6 h (migration assay) or 24 h (invasion assay), cells on the upper surface of the filter were wiped off with cotton swabs. Cells on the lower surface of the filter were fixed with methanol and stained with hematoxylin and eosin. More than six views/insert were analyzed under a light microscope (200×), and stained cells were counted. Data represent mean ± standard deviation of three independent experiments.
8
Cell Migration and Wound Healing Assays
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For transwell migration assay (BD Biosciences), RAW264.7 cells resuspended in serum‐free medium were pretreated with various concentrations of DSPS. Cells were then added to insert chambers and incubated for 16 hours. Cells on the upper surface of the inserts were completely wiped off with a cotton swab, while cells that migrated to the underside of the inserts were fixed with 4% paraformaldehyde for 15 minutes at room temperature. Cells were then stained with 0.5% crystal violet in 20% methanol for 30 minutes, washed three times with distilled water, and visualized with a Leica stereomicroscope (Leica Microsystems, Wetzlar, Germany).47 , 48 For wound healing assay, cells were grown to 90% confluence in a 12‐well plate, and a scratch wound was created in the monolayer using a pipette tip. Cell debris was removed by washing three times with phosphate‐buffered saline (PBS). Cells were incubated in complete culture medium for the indicated times to allow wound healing. Phase‐contrast images of the wound were captured at the indicated time points.
9
Chemotactic Motility of HUVECs
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The chemotactic motility of HUVECs was determined using a transwell migration assay (BD Biosciences) with 6.5 mm diameter polycarbonate filters (8 mm pore size). Briefly, the RPMI 1640 medium containing 10 ng/mL VEGF (500 μL) was placed into the bottom chambers. HUVECs (5 × 104) were incubated with MC-LR (0–40 μM) in RPMI 1640 medium containing 0.5% FBS for 3 hours at 37°C before seeding into the upper chambers. After 8 h of incubation, nonmigrated cells in the upper chamber were removed. The migrated cells were fixed with 4% paraformaldehyde and stained with 1% calcein-AM. Images were taken using an Olympus microscope. Migrated cells in four random fields were quantified by manual counting. Three independent experiments were performed.
10
Cell Migration and Wound-Healing Assays
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For the Transwell migration assay, 8 μm pores (BD Bio-sciences, Franklin Lakes, NJ, USA) were used in 24-well plates to evaluate migration ability. SACC cells were serum starved overnight and 1×105 cells were added onto the upper inserts in 200 μL serum-free RPMI 1640 media. The lower chamber was added with 600 μL 1% serum media as a chemoattractant. The inserts were removed after 24 hours and nonmigrating cells on the upper surface of the inserts were wiped with a cotton swab. Migrated cells on the undersurface of the membrane were fixed in 4% paraformaldehyde for 10 minutes and then stained with crystal violet staining solution (Beyotime, Shanghai, China) for 10 minutes. Quantification was performed by counting the migrated cells in five randomly selected high-power fields (200×).
For wound-healing assay, cells were grown on six-well plates to about 80% confluence. A scratch was generated with a 200 μL pipette tip. Subsequently, the wounded monolayers were washed with PBS three times to remove nonadherent cells, and then 2 mL culture medium with 1% serum was added with the indicated agents for 36 hours. Wound healing was quantified and photographed. The wound-healing assay was performed in triplicate.
For wound-healing assay, cells were grown on six-well plates to about 80% confluence. A scratch was generated with a 200 μL pipette tip. Subsequently, the wounded monolayers were washed with PBS three times to remove nonadherent cells, and then 2 mL culture medium with 1% serum was added with the indicated agents for 36 hours. Wound healing was quantified and photographed. The wound-healing assay was performed in triplicate.
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