In the migration assay, transfected CAL27 cells were inoculated in the upper chamber of the Transwell system (BD Biosciences, San Jose, CA, United States), and the lower chamber was filled with 500 μl of medium containing 10% FBS. 24 h later, the cells remaining on the surface of the filter membrane were gently wiped off with a cotton swab, and the cells passing through the membrane were fixed with methanol and then stained with crystal violet solution. Under an inverted microscope, three randomly selected fields of view (including the center and periphery of the membrane) were used to count the number of cells. Matrigel (BD Biosciences, San Jose, CA, United States) was used to coat the filters of the Transwell system in the invasion assay, and the other steps were the same as for the migration assay.
Transwell system
Manufactured by BD
Sourced in United States
The Transwell system is a laboratory equipment used for cell culture studies. It consists of a permeable membrane insert that is placed inside a well or plate, allowing for the separation of different cell types or compartments within a single culture environment.
Automatically generated - may contain errors
Lab products found in correlation
Matrigel, by BD (21 mentions)
Biocoat matrigel invasion chamber, by BD (4 mentions)
Crystal violet, by Merck Group (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Ckx41, by Olympus (2 mentions)
Matrigel, by Merck Group (2 mentions)
Giemsa s azur eosin methylene blue solution, by Merck Group (2 mentions)
Methylene blue, by Merck Group (2 mentions)
Ebm 2, by Lonza (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions)
Fortessa x20, by BD (1 mentions)
Matrigel coated insert, by BD (1 mentions)
Infinite 200 pro, by Tecan (1 mentions)
Matrigel, by Corning (1 mentions)
Collagen type 1, by Corning (1 mentions)
Type 4 collagen, by Corning (1 mentions)
Dmem with high glucose, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Cck 8 reagent, by Dingguo (1 mentions)
Fibronectin, by Merck Group (1 mentions)
82 protocols using transwell system
1
Transwell Assay for Cell Migration and Invasion
2
Quantifying Cell Invasion using Transwell Assay
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Cell invasion assays were performed using a Transwell system (Falcon; Corning Life Sciences) as previously described (14 (link)). Briefly, Matrigel inserts containing an 8 µm pore size membrane with a thin layer of Matrigel Basement Membrane Matrix were pre-incubated with serum-free media for 2 h at 37°C. A total of 105 cells were then plated in the top chamber which contained a Matrigel (250 µg/ml)-coated membrane. In the assay, cells were plated in medium without serum or growth factor, and medium supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) was placed in the lower chamber as a chemoattractant. After incubation for 36 h at 37°C in a humidified incubator with 5% CO2. The cells of the membrane were fixed using 3.7% formaldehyde and stained using 0.1% crystal violet (Sigma-Aldrich; Merck KGaA) at room temperature for 30 min. Cells that had not infiltrated the membrane pores were removed with a cotton swab. Images of invaded cells on the lower surface of the membrane were observed using a Ti-U bright field microscope (Nikon Corporation). In each condition, 5 independent fields were quantified using ImageJ2 (National Institutes of Health) and the average of these fields considered as the mean number of invasive cells per condition.
3
Neural Stem Cell Migration Assay
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NSCs were seeded apically on laminin-coated
inserts of the trans-well system (8 μm pore size, Falcon, Corning,
USA) and incubated for 24 h in a NSC-maintaining medium. Afterward,
the basolateral medium was replaced by U87 or a NHA-conditioned medium.
As controls, basolateral media and NSC-maintaining media were used
with or without supplementary growth factors. After 12, 24, and 48
h, the insets were washed with DPBS, and the apically growing NSCs
were gently removed using cotton swabs. The insert membranes were
cut out and coated with a DAPI-containing mounting medium. The cell
number was determined using fluorescence microscopy. To investigate
the impact of irradiated cells, the NSCs were incubated in medium
collected from U87 and NHA cells after treatment with [125I]ITdU (100 kBq for 24 h). To determine the possible effect of NGs
on migration potential, the NSCs were incubated with NGs as described
above prior to seeding on inserts of the trans-well system.
inserts of the trans-well system (8 μm pore size, Falcon, Corning,
USA) and incubated for 24 h in a NSC-maintaining medium. Afterward,
the basolateral medium was replaced by U87 or a NHA-conditioned medium.
As controls, basolateral media and NSC-maintaining media were used
with or without supplementary growth factors. After 12, 24, and 48
h, the insets were washed with DPBS, and the apically growing NSCs
were gently removed using cotton swabs. The insert membranes were
cut out and coated with a DAPI-containing mounting medium. The cell
number was determined using fluorescence microscopy. To investigate
the impact of irradiated cells, the NSCs were incubated in medium
collected from U87 and NHA cells after treatment with [125I]ITdU (100 kBq for 24 h). To determine the possible effect of NGs
on migration potential, the NSCs were incubated with NGs as described
above prior to seeding on inserts of the trans-well system.
4
Human BBB Transmigration Assay
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The human blood-brain-barrier (BBB) endothelial cell line HCMEC/D3 is an immortalized endothelial cell line derived from a primary cell culture coexpressing hTERT and the SV40 large T antigen via a lentiviral vector system [25 (link)]. Transmigration assays were performed using the Transwell system (8.0 μm pore filters; BD Falcon, France) as previously described [26 (link)]. Cell concentration was tested and concentrations which allowed more than 5% human albumin diffusion in the lower chamber after 6 hours were discarded. Two days before the migration assay, 1 × 106 HCMEC/D3 cells were cultured on the apical side of the filter insert. All cells were grown in endothelial basal medium-2 (EBM-2, Lonza) supplemented with human serum (PAA). 5 × 105 B cells were added on top of the BBB layer. After 18 h at 37°C with 5% CO2, the contents of the bottom chamber were collected with EDTA to detach any adherent cell, and B cells were counted. The transmigrated cells were washed, stained with CD19-PE, and incubated with albumin or MOG coupled beads as indicated previously.
5
Transwell Assay for Cell Migration
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Transwell assays were performed to determine the migratory ability of the various types of SKOV3 cells transfected with SLIT2 overexpression or knockdown plasmids, as aforementioned. The Transwell system (Falcon; Corning Life Sciences) consisted of upper and lower chambers. The cells seeded in the upper chamber were able to migrate through the membrane to the lower chamber. In total, 3×105 cells were seeded into the upper chamber in serum-free RPMI-1640 medium and the lower chamber was filled with RPMI-1640 medium with 10% FBS. After incubation for 24 h at 37°C, the cells that were attached to the reverse side of the Transwell membrane were stained using crystal violet for 30 min, and total cells were counted under a light microscope at ×100 magnification. In total, three independent experiments were performed for each cell type.
6
Preparation of Dermo-Epidermal Skin Analogues
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Preparation of the dermo-epidermal skin analogues in six-well cell culture inserts with 3.0 μm pore size membranes, in a transwell system (BD Falcon, Switzerland), was performed as described in Biedermann et al. (Biedermann et al., 2015 (link)). In brief, the corresponding dermal compartment consisting of a membrane covered with collagen type I hydrogel containing human fetal fibroblasts was prepared first. Therefore, collagen type I (Symatese, France) was mixed with neutralization buffer containing NaOH and 1 × 105 fibroblasts (passage 2) for polymerization. Those dermal analogues were further cultivated for 7 days in DMEM containing 10% FCS. After cultivation, 5 × 105 keratinocytes (passage 2) were distributed onto each dermal equivalent. After one additional week of cultivation in SFM (Invitrogen, Switzerland), with medium change every second day, the dermo-epidermal skin substitutes were ready for transplantation.
7
Transwell Migration Assay for hBMSCs and Chondrocytes
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To investigate the effect of sEVs on migration of hBMSCs and chondrocytes, transwell migration assay was performed as previously described [31 (link)]. Briefly, 10 × 104 cells cultured in serum-free medium were added to the upper chamber in transwell system (BD falcon, USA) and migrated for 8 h. The cells on the upper chamber were removed and the remaining cells on the undersurface of the transwell were fixed using 4% PFA. After be stained using 0.2% crystal violet, the migrated cells were visualized under the microscopy. Data were presented as mean ± SD of three number of replicates.
8
Cell Migration and Invasion Assay
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Transwell assays were performed in order to determine the migration and invasion ability of the CTNNBIP1 overexpression cells and the vector control cells. A similar analysis was also performed using CTNNBIP1 knockdown cells and si-control cells. The transwell system (Falcon, Bedford, MA, USA) consisted of upper and lower chambers, separated by a layer of millipore membrane with pore size of 8 μm. The cells seeded in the upper chamber could migrate through the membrane to the lower chamber. About 5 × 105 cells were seeded into the upper chamber of the transwell with a serum-free DMEM medium, and the lower chamber containing a 10% FBS/DMEM attractant medium. After incubation for 24 h, the cells attached to the reverse phase of the membrane were stained by crystal violet, or were observed for cells with fluorescence, and were counted under a microscope in six randomly selected fields. The experiment was carried out three times in order to reduce the possible effects of biological variability.
9
T cell suppression and MDSC migration
10
Transwell-Based Migration Assay for BMSCs
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According to the established protocol [33] , transwell migration assay was implemented to evaluate the effect of sEVs on migration of BMSCs. In brief, BMSCs incubated in the medium without serum were seeded the upper chamber of transwell system (BD falcon, USA). After 8 h migration, BMSCs were xed by 4% PFA. Then, 0.2% crystal violet was used to stain the migrated cells, and the cells on the upper chamber were removed. The microscopy was used to visualize the migrated BMSCs.
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