Matrigel cell invasion assays were performed using 8‐μm polycarbonate membranes (Corning, Acton, MA, USA) according to the manufacturer's instructions. Cell suspension (5 × 105 cells; 100 μl) was added to the upper chamber, while the lower chamber was filled with RPMI‐1640 medium containing 10% foetal calf serum. The plates were incubated for 24 hrs, and then cells that had invaded to the lower membrane surface were fixed in methanol for 30 min. and stained with haematoxylin (Sigma). For each filter, the numbers of cells that invaded to the lower surface of the membrane were counted in five randomly selected fields of view at 200 × magnification using a Nikon E200 light microscope (Nikon, Melville, NY, USA). Mean cell numbers were calculated from the data obtained from each experiment repeated three times.
8 μm polycarbonate membrane
Manufactured by Corning
Sourced in United States
The 8 μm polycarbonate membrane is a laboratory equipment product designed for filtration applications. It has a pore size of 8 micrometers and is made of polycarbonate material. The core function of this membrane is to facilitate the separation and filtration of various samples and solutions.
Automatically generated - may contain errors
Lab products found in correlation
E200 light microscope, by Nikon (1 mentions)
Haematoxylin, by Merck Group (1 mentions)
Sdf 1α, by Thermo Fisher Scientific (1 mentions)
Cyquant kit, by Thermo Fisher Scientific (1 mentions)
Ab39250, by Abcam (1 mentions)
Geltrex matrix, by Thermo Fisher Scientific (1 mentions)
Cell counter, by Countstar (1 mentions)
Matrigel coated, by Corning (1 mentions)
Precoated matrigel, by Corning (1 mentions)
9 protocols using 8 μm polycarbonate membrane
1
Matrigel Cell Invasion Assay Protocol
2
Transwell Assay for Cell Migration
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Cell migration ability was assessed by Transwell assay. 1 × 105 cells were plated in the upper chamber of a 24-well plate with 8-μm polycarbonate membranes (Corning) filled with 200 μL serum-free DMEM medium. 600 μL DMEM containing 10 % FBS (as a chemoattractant) was added to the lower chamber. After 24 h of incubation at 37 °C, 5 % CO2, the cells remaining in the upper chamber were removed with a cotton swab, and cells migrated to the lower surface were fixed with 4 % paraformaldehyde for 30 min and then stained with 0.1 % crystal violet for 20 min. Cells were counted under a microscope (x200) in five random visual fields. Each experiment was independently repeated three times.
3
Transwell Assay for CD34+ HSPC Migration
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The transwell cell migration assay was performed as follows: HUVEC at a density of 1 × 104 cells in endothelial cell growth medium were plated in the upper chamber of 24-well culture plates with 8 μm polycarbonate membranes (Corning Inc.) that were coated with 0.05% gelatine (Biochrom, Berlin, Germany). The endothelial cells were cultured until a confluent adherent monolayer was reached. Then the adherent cell layer and 5 × 104 CD34 + HSPC could be treated with 10 μg/ml of antibodies in SFEM without cytokines for 30 min at 37°C and 5% CO2. The medium was removed from the adherent cell layer and the inserts were placed in a 24 well plate. Then, 5 × 104 CD34 + HSPC suspension treated with or without antibodies was pipetted onto the upper chamber. The lower chamber was filled with SFEM with or without 100 ng/ml stromal cell-derived factor 1α (SDF-1α; PeproTech, Hamburg, Germany). Without SDF-1α the lower chambers were considered as negative control, whereas those containing SDF-1α acted as positive control. The CD34 + HSPC were allowed to migrate for 16 h at 37°C and 5% CO2. The number of migrated cells in the lower chamber was determined measuring the DNA content with the CyQuant kit (Invitrogen).
4
Transwell Migration of hMSCs by MSC-Exo
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The hMSCs (5 × 105 cells/cm2) in DMEM(-) were dispersed within the upper chamber of transwell dishes with 8 μm polycarbonate membranes (Corning Inc., NY, USA). MSC-Exo (5 μg/mL) + DMEM(-) (MSC-Exo), MSC-Exo (5 μg/mL) + anti-VEGFA antibody (100 ng/mL; ab39250, Abcam PLC, Cambridge, UK) + DMEM(-) (Exo-antiVEGF), MSC-CM, or DMEM(-) were placed on the lower chamber. After 48 hours of incubation, the surface of the membrane was rinsed with phosphate-buffered saline (PBS) and wiped with a cotton bud. The membrane was then stained with hematoxylin and the total number of cells that migrated was counted.
5
Transwell Invasion Assay for Cell Motility Analysis
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Transwell chambers with 24 wells and an 8 μm polycarbonate membrane (Corning, NY, USA) were used, following the manufacturer’s protocol. The upper chambers were coated with 100 µL of DMEM-diluted Geltrex matrix (Gibco) and incubated at 37 °C for 6 h to allow the gel to solidify. The cultured cells were detached using 0.25% trypsin-EDTA solution and counted in a Neubauer chamber. Then, 1 × 105 cells were seeded into the upper chambers in 200 µL of serum-free media. A total of 500 µL of complete medium was added to the lower chamber as a chemo attractant. After 12 h, the cells remaining in the upper side of the polycarbonate membrane were removed with cotton swabs. Bottom chambers containing invasive cells were washed (twice with PBS), fixed in 100% methanol, and stained with DAPI (5 μm) for 5 min. Ten visual fields of each insert were randomly chosen, and photographed at 4x magnification. The number of cells/fields was quantified using ImageJ software (NIH, Bethesda, MD, USA).
6
Chemotaxis Assay for Dendritic Cells
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Chemotaxis of fmDCs or cryoim-mDCs was assessed by their migration through an 8 μm polycarbonate membrane (Corning, NY, USA) using CCL19 as the chemoattractant. DCs were seeded (5 × 104 cells in 100 μL of RPMI 1640) in the upper chamber in 24-well cell culture plates and the lower chamber contained 500 μL of RPMI 1640 with or without CCL19 (final concentration, 500 ng/mL). Each condition was set up in three repeats. The plates were incubated for 4 h at 37 °C in 5% CO2. The cells remaining in the upper chamber were removed and the migrated cells were harvested by washing the bottom side of the inserts and the wells 3 times. The total migrated cells were counted by use of Cell Counter (IC1000, Countstar, Shanghai, China) and the results were expressed as the mean number of migrating cells ± standard deviation (SD).
7
Transwell Assay for Cell Migration and Invasion
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Migration and invasion experiments were performed by transwell assay. For migration assays, transfected cells were seeded at a density of 1 × 105 cells in 24‐well transwell chambers with 8‐μm polycarbonate membrane (Corning, New York, USA), and 100 μl serum‐free medium was added to the upper chamber. Next, 600 μl medium containing 30% fetal bovine serum was added to the lower chamber and the cells were cultured at 37°C for 18 h for migration. After the cells on the upper surface of the filter were removed with cotton swabs, the migrating cells attached to the lower surface of the membrane were fixed with 4% paraformaldehyde solution, stained with 0.5% crystal violet, and counted in nine random fields under the microscope. For invasion assays, a Matrigel coating was added to the polycarbonate membrane. Three independent experiments were performed.
8
Transwell Invasion Assay for Cell Migration
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The transwell invasion assay was performed in 24-well plates with a 6.5mm insert transwell chamber with 8μm polycarbonate membrane (Corning) pre-coated Matrigel (Corning). The single cell suspension was added into to upper chamber with 5*104 cells in 200μl culture medium with 2% FBS, and 500μl culture medium with 20% FBS was added into the lower chamber. After 48 h, discarded the solution in the upper chamber and wiped the upper layer of the membrane. Move the chamber into 4% PFA to fix for 5 min. Stain the membrane with crystal violet for 5 min. Finally, obtained the photographs on the microscope. All experiments were performed in triplicate.
9
Transwell Invasion Assay for Hypoxia
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The transwell invasion assay was performed in 24-well plates with a 6.5 mm insert transwell chamber with 8 μm polycarbonate membrane (Corning) pre-coated Matrigel (Corning). The single cell suspension was added into upper chamber with 5×104 cells in 200 μL culture medium with 2% FBS, and 500 μL culture medium with 20% FBS were added into the lower chamber. After incubating in a hypoxic incubator with 1% O2 and 5% CO2 for 48 to 72 h, discarded the solution in the upper chamber and wiped the upper layer of the membrane. Move the chamber into 4% PFA to fix for 5 min. Stain the membrane with crystal violet for 5 min. Finally, we obtained the photographs on the microscope. All experiments were performed in triplicate.
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