THP-1 (2 × 105) or U937 (2 × 105) cells were seeded inside hanging inserts (Millicell® hanging inserts, 8.0 μm, Merck) suspended over wells with conditioned media in a 12-well plate. The conditioned media were obtained from 5 × 105 pancreatic cancer cells (AsPC-1, HPAF-II or PANC-1 cells) cocultured with 5 × 105 PSCs in a Millicell® hanging insert (0.4 μm polyethylene terephthalate membrane, Merck) in 6-well plates for 24 h. The conditioned media were centrifuged at 1000 g for 5 min before being employed in further experiments. The migrated cells were counted in four different fields, and the experiments were repeated in triplicate. Primary human monocytes (2.5 × 105) were seeded inside hanging inserts (8.0 μm, Merck) suspended in the wells with or without S100A9 stimulation in a 24-well plate.
Millicell hanging insert
Manufactured by Merck Group
Sourced in Germany
Millicell hanging inserts are a type of lab equipment used for cell culture applications. They provide a permeable membrane support that allows for the exchange of nutrients, gases, and other substances between the upper and lower chambers of a cell culture system.
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12 protocols using millicell hanging insert
1
Chemotaxis Assay for Monocytes and Pancreatic Cancer Cells
2
Pancreatic Cancer Cells Promote Leukocyte Migration
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THP-1 (2x10 5 ) or U937 (2x10 5 ) cells were seeded inside hanging inserts (Milli-cell® hanging inserts, 8 µm Merck) suspended over wells with conditioned media in a 12-well plate. The conditioned media were obtained from 5x10 5 pancreatic cancer cells (AsPC-1, HPAF-II or PANC-1 cells) cocultured with 5x10 5 PSCs in a Millicell® hanging insert (0.4 µm polyethylene terephthalate membrane, Merck) in 6-well plates for 24 hours. The conditioned media were centrifuged at 1000 g for 5 minutes before being employed in further experiments. The migrated cells were counted in four different fields, and the experiments were repeated in triplicate.
3
Bacterial Growth Profiling on Carbon Sources
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Aliquots (50 μl) of the co-isolated bacteria from the overnight cultures, were inoculated into fresh MH medium with shaking to achieve the cell density of OD600: 0.6, respectively. The KP6870155 was then inoculated into Millicell hanging insert (Merck) containing a sole carbon source. The insert only allowed the passage of small-secreted metabolites but not bacterial cells. AB6870155 was directly inoculated into 6-well plates containing the single carbon source that cannot be used by it and subsequently combined with inserts containing KP6870155. The assembled 6 well plates were incubated at 37 °C for 24 h with shaking. Fluorostar Omega spectrometer (BMG Labtech) was used to determine the growth of AB6870155. Each carbon source was assessed in triplicates.
4
Co-culture of Glioblastoma Stem Cells
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The recipient normal cells of the brain were seeded in 24-well plates, as described above. One day later, small GBM8 neurospheres grown in GSC conditions were transferred to the upper chamber of the Millicell hanging insert with 1.0 μm pore size (Millipore). For co-cultures of GSCs with neurons, the optimized media consisted of Neurobasal, 1× B-27, 0.5× N-2 supplement, 0.5 mM GlutaMAX, and 1% Antibiotic-Antimycotic Solution. For co-cultures of GSCs with glia, the optimized media consisted of DMEM-F12, 1× B-27, 0.5× N-2 supplement, and 1% Antibiotic-Antimycotic Solution. For co-cultures of GSCs with endothelial cells, the optimized media consisted of Mouse Endothelial Cell Basal Medium (Cell Biologics), 1× B-27, 0.5× N-2, 0.1% VEGF, 0.1% ECGS, 0.1% EGF, 0.1% Hydrocortisone, 2 mM L -Glutamine and 1% Antibiotic-Antimycotic Solution.
5
Migration and Invasion Assay Protocol
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Migration and invasion assays were performed in 24-well Millicell hanging insert (Millipore) or 24-well BioCoat Matrigel Invasion Chambers (BD Biosciences). For the invasion assay, cells were starved with serum-free medium for 24 hours before the assay. A total of 5 Â 10 4 cells were seeded inside the top chamber with serum-free medium, whereas medium supplemented with 10% FBS was filled into the bottom chamber that served as the chemoattractant. After 48-or 72-hour incubation, cells that migrated or invaded through the membrane (migration) or Matrigel (invasion) were fixed, stained with Crystal Violet, air dried, and mounted on the slides. The number of cells were counted in 10 fields and imaged using SPOT imaging software (Nikon). Data are expressed as the mean AE SD of triplicate wells within the same experiment.
6
Cell Migration Assay
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105 cells resuspended in serum free DMEM were added to the top of 24-well Millicell hanging inserts (Millipore). As a chemoattractant, 0.75 ml DMEM-10% FBS was added to the bottom chamber. After 24 hours of incubation at 370C, cells that migrated or invaded through the membrane (migration) were fixed with 70% ethanol and stained with 0.2% crystal violet in 2% ethanol for 10 min. The inserts were washed extensively with distilled water and air-dried. The number of migrated cells were measured using ImageJ software.
7
Invasion and Migration Assay Protocol
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Invasion and migration assays were conducted in 24-well BioCoat Matrigel Invasion Chambers (BD Biosciences) or 24-well Millicell hanging inserts (Millipore). Cells re-suspended in serum free DMEM were added to the top chamber and DMEM supplemented with 10% FBS was added to the bottom chamber as a chemoattractant. After 48 hrs incubation at 37°C, the number of cells that invaded through the Matrigel (invasion) or membrane (migration) was counted in 10 fields under 4x objective lens and imaged using SPOT imaging software (Nikon, Japan).
8
Bioengineered Skin Tissue Construction
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The epidermis was removed from pieces of surgical waste skin and discarded. The dermis was subjected to repeated freeze-thaw cycles followed by gamma irradiation, as described previously (Philippeos et al., 2018 (link)). De-epidermised dermis (DED) was placed in six-well microplate Millicell hanging inserts (Millipore). 5 × 105 NHDF were resuspended in 30 μl complete DMEM and injected into the upper dermis using a 0.3 ml insulin syringe and needle (Becton Dickinson). NHDF-containing DED was cultured submerged in 3 ml/well complete DMEM for 24 h. 1 × 106 keratinocytes (strain Km) were resuspended in 30 μl complete FAD, seeded onto the DED, and cultured in 2 ml/well complete FAD above the air–liquid interface for 2 weeks. The histological analysis is described in Supplementary Materials and Methods .
9
Monocytic Migration Potential under Iron Stimulation
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To analyse monocytic migration potential, isolated monocytes were stimulated with i.v. iron preparations for 3 h at 37°C (5 × 105 monocytes per condition), washed twice in RPMI 1640 and labelled with anti-CD45 antibody (Supplementary data, Table S1 ) for 1 h at 37°C. Cells were seeded into the upper chamber of Millicell hanging inserts (8 µM pore size; Millipore, Schwalbach, Germany), which were placed in 24-well plates. Lower chambers were filled with RPMI 1640 enriched with 50 ng/mL monocyte chemotactic protein-1 (MCP-1; Biolegend, Fell, Germany). After 60 min at 37°C, the number of transmigrated cells was evaluated by fluorescence microscopy in 10 microscopic fields per sample.
10
Transwell Invasion and Migration Assay
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The invasion and migration assays were performed in 24-well Millicell hanging inserts (Millipore) with or without a Matrigel layer (BD Biosciences) according to the manufacturer's instructions. Briefly, 1 × 105 cells were seeded into the top chamber, and DMEM with 10 % FBS was added to the bottom chamber as a chemoattractant. After a 48 hour incubation at 37 °C, the numbers of cells that invaded the Matrigel (invasion) or membrane (migration) were counted in 10 fields using a 40× objective lens.
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