The in vitro migration assay was performed in the transwell inserts, with an 8-μm pore size, the membrane of which was uncoated (SPL, Germany). The cells were treated by PBS (Phosphate buffered saline) with 10-100 or 500 nM kp-10 (Anaspec, USA) for 24 hours and one other group of cells was not treated with kp-10. Then the cells were trypsinized and a number of 2 × 104 mesenchymal stem cells in 200 μl serum-free medium were seeded into the top chamber. Then, 30% of Fetal calf serum (FCS) or 4 × 104 melanoma cells in 500 μl medium were added to the lower chamber as a chemoattractant. After 24 hours of incubation at 37˚C, the non-migrated cells were removed from the top of the membrane, whereas, cells on the bottom of the membrane were trypsinized with 300 μl of trypsin and were suspended into the medium and counted in five minutes by flow cytometry (BD Calibur™ (Becton Dickinson)) after obtaining the appropriate gate. Results were obtained by three experiments.[23 (link)24 (link)]
Transwell inserts
Manufactured by SPL Life Sciences
Sourced in United States
Transwell inserts are a type of cell culture insert used for in vitro studies of cell migration, invasion, and permeability. They consist of a porous membrane that separates two compartments, allowing for the study of interactions between cells or the passage of substances across the membrane.
Automatically generated - may contain errors
Lab products found in correlation
Lipopolysaccharides (lps), by Merck Group (3 mentions)
Facsaria 2, by BD (2 mentions)
Calibur, by BD (1 mentions)
Kp 10, by AnaSpec (1 mentions)
Caerulein, by Merck Group (1 mentions)
Dg250 anaerobic workstation, by Don Whitley Scientific (1 mentions)
6 well plate, by SPL Life Sciences (1 mentions)
Geltrex, by Thermo Fisher Scientific (1 mentions)
Epoch microplate spectrophotometer, by Agilent Technologies (1 mentions)
14 protocols using transwell inserts
1
In Vitro Mesenchymal Stem Cell Migration Assay
2
Caerulein and LPS-Induced Pancreatic Acinar Cell Co-Culture
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All co-culture experiments were conducted in six-well cell culture plates containing 0.4-μm pore-sized transwell inserts (SPL Life Science, Pocheon, Korea). A total of 3 × 105 hAT-MSCs, scr siRNA-transfected hAT-MSCs, or TSG-6 siRNA-transfected hAT-MSCs were plated at the bottom of the six-well plates (SPL Life Science). After the attachment of cells to the plates, isolated PACs were seeded onto the transwell inserts and stimulated with 100 nM caerulein (Sigma-Aldrich) and 10 mg/mL LPS (Sigma-Aldrich) for 12 h. The co-culture system was maintained at 37 °C in a humidified atmosphere with 5% CO2. Naive PACs and non-hAT-MSCs treated with caerulein and LPS served as the naive and positive control groups, respectively.
3
Oxygen-Deprived iPSC-Myoblast Co-Culture
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The iPSCs were cultured in mTeSR1 medium in Transwell inserts (SPL, Korea) coated with Matrigel matrix, and the cells were allowed to reach 70–80% confluence for co-culture. The C2C12 myoblasts were cultured with growth medium or differentiation medium in 6-well plates. The day before co-culture, the medium was exposed to a DG250 anaerobic workstation (Don Whitley Scientific, UK) containing a 5% CO2, 10% H2, and 85% N2 gas mixture for oxygen removal. The iPSCs were washed three times with phosphate buffered saline (PBS, BI, USA) and co-cultured with C2C12 myoblasts or C2C12 myotubes together in a Transwell inserts system (6-well plates) in a DG250 anaerobic workstation at 37 °C for 24 h of oxygen deprivation. Further, 1.5 mL of the medium was added to the Transwell insert and 2.5 mL of the medium was added to the 6-well plates.
4
Microcarrier-based Co-culture of UCB-CD34+ Cells and MSCs
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The UCB-CD34+ cells were cultured in a co-culture with the feeder cell
UCB-MSCs in the presence and absence of microcarrier beads for 7 days. In the co-culture
experiments, 1×105 UCB-MSCs and UCB-CD34+ cells were cultured on the
microcarrier beads for seven days at 37°C and 5% CO2 . To address the
importance of cellcell interactions between the UCB-CD34+ cells and UCBMSCs, we
used 0.4 µm pore size transwell inserts (6.5 mm diameter, 0.4 µm pore size, SPL Life
Sciences, Seoul, Korea) in our co-culture groups. The culture groups used in this study
were classified into five groups: A. Freshly isolated CD34+ cells, B.
CD34+ cells co-cultured with a monolayer of UCB-MSCs, C. CD34+cells co-cultured with UCB-MSCs expanded on the microcarrier beads, D. CD34+cells and UCB-MSC as a non-contact co-culture using transwell and E. CD34+
cells and UCB-MSCs expanded on microcarrier beads as a non-contact co-culture using
0.4 µm pore size transwell inserts. Figure 1 illustrates the schematic diagrams of the
culture conditions. In further functional studies, UCB-MSCs and CD34+ cells
were easily separated by sedimentation due to the distinct weight differences between
microcarriers containing adherent MSCs and the suspended CD34+ cells.
UCB-MSCs in the presence and absence of microcarrier beads for 7 days. In the co-culture
experiments, 1×105 UCB-MSCs and UCB-CD34+ cells were cultured on the
microcarrier beads for seven days at 37°C and 5% CO2 . To address the
importance of cellcell interactions between the UCB-CD34+ cells and UCBMSCs, we
used 0.4 µm pore size transwell inserts (6.5 mm diameter, 0.4 µm pore size, SPL Life
Sciences, Seoul, Korea) in our co-culture groups. The culture groups used in this study
were classified into five groups: A. Freshly isolated CD34+ cells, B.
CD34+ cells co-cultured with a monolayer of UCB-MSCs, C. CD34+cells co-cultured with UCB-MSCs expanded on the microcarrier beads, D. CD34+cells and UCB-MSC as a non-contact co-culture using transwell and E. CD34+
cells and UCB-MSCs expanded on microcarrier beads as a non-contact co-culture using
0.4 µm pore size transwell inserts. Figure 1 illustrates the schematic diagrams of the
culture conditions. In further functional studies, UCB-MSCs and CD34+ cells
were easily separated by sedimentation due to the distinct weight differences between
microcarriers containing adherent MSCs and the suspended CD34+ cells.
5
LPS-stimulated Macrophage-MSC Interaction
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After cPBMC-derived macrophages were stimulated with 200 ng/ml lipopolysaccharide (LPS; Sigma-Aldrich, St. Louis, MO, USA) for 24 h, the LPS-stimulated macrophages were plated at a density of 2 × 105 cells per well in 24-well plates. Subsequently, 2 × 104 cAT-MSCs, control siRNA-cAT-MSCs, or TSG-6 siRNA-cAT-MSCs were seeded onto 0.4-μm pore-sized Transwell inserts (SPL Life Science, Pocheon, Korea) and incubated for 48 h. The macrophages were harvested for further experiments.
6
Intestinal Epithelial Monolayer Absorption
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The monoculture system of Caco-2 cells, representing the intestinal epithelium monolayers of tight junctions, was prepared as follow; after coating Transwell® inserts (SPL Life Science, Gyeonggi-do, South Korea) with MatrigelTM matrix (Becton Dickinson, Bedford, MA, USA) for 1 h, supernatants were removed, and then inserts were washed with DMEM. Caco-2 cells (4.5 × 105 cells/well) were grown on upper insert sides, cultured for 21 days, and treated with SunActiveTM products or an equivalent amount of FePP or ZnO-NPs (50 μg/ml Fe or Zn content) for 6 h. The concentrations of transported Fe or Zn in basolateral solutions were determined by ICP-AES (JY2000 Ultrace, HORIBA Jobin Yvon).
7
Anticancer Drug Cytotoxicity Assay
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TCs were plated at a density of 5 × 106 cells per well in 6-well plates (SPL Life Science, Korea), and 5 × 105 SCs grown in media containing an anticancer drug (DOX; dose, 0, 0.005, 0.05, and 0.5 µg/mL) were seeded onto 0.4-µm pore-sized Transwell inserts (SPL Life Science) for 48 h.
8
Stem Cell Osteogenesis Influenced by Endothelial Cells
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To evaluate the osteogenic responses of stem cells to endothelial cells and spheroids, hASCs (1 × 105 cells/cm2) were seeded onto 6‐well tissue culture plates. The One‐SP‐spheroids‐based dECM construct (containing 56 spheroids) was placed onto the top level of transwell inserts (SPL Life Science, USA). As controls, 2D cultured isolated HUVECs and conventionally prepared HUVEC‐spheroids (C‐Spheroids) were positioned on the top level in a dECM construct. The GM was changed every 2 days, and the cells were cultured at 37°C and 5% CO2.
9
HCEC-Macrophage Co-culture Protocol
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HCECs were seeded in 6 well-plates at a density of 1.5 × 105 cells/well and cultured for 24 h. Co-culture were conducted using transwell inserts with a pore size of 0.4 μm (SPL Life Sciences, Pocheon-si, South Korea) placed into 6-well plates. M1 macrophages were resuspended in RPMI 1640 at density of 1 × 105 cells/insert and then treated with EGF. The co-cultures of M1 macrophages and HCECs were incubated undisturbed at 37 °C in a humidified 5 % CO2 incubator for 24 h. RNA was subsequently extracted from HCECs (6-well plate) for analysis.
10
Pramlintide Effects on Endothelial Cells
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Pramlintide was provided by AstraZeneca Co. (Cambridge, UK). Dulbecco’s modified eagle’s medium (DMEM), fetal bovine serum (FBS), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay kit were purchased from Bioidea Co. (Tehran, I.R. Iran). Transwell® inserts were obtained from SPL Life Sciences Co., Ltd. (Gyeonggi-do, Korea). Geltrex was purchased from Gibco- BRL, Life Technologies Inc. (California, USA). Calcein acetoxymethyl dye was prepared from Santa Cruz Biotechnology Inc. (Santa Cruz, Canada). Recombinant human vascular endothelial growth factor (VEGF) and the enzyme-linked immunosorbent assay (ELISA) kit for its evaluation were purchased from PeproTech, Inc. (New Jersey, USA).
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