Chemotaxis assays with human ECs were performed in a Boyden chamber, as previously described36 . In brief, PVP-free polycarbonate filters (8 μm pore size; Neuroprobe) were coated with 1% gelatin for 2 h at 37° C. A total of 10 ng/ml VEGF-A dissolved in serum-free medium was seeded in the lower compartment of the chamber; cells were serum starved overnight, and then suspended in serum-free medium at a concentration of 2.5 × 106 cells/ml, and 50 μl of the suspension was added to the upper compartment. After 5 h of incubation at 37° C with 5% CO2, the upper surface of the filters was scraped with a rubber policeman, and the filters were fixed and stained with Diff-Quick (Dade Behring). Four random fields of each sample in the lower surface of the filters were counted at ×10 magnification.
Pvp free polycarbonate filters
Manufactured by Neuro Probe
Sourced in United States
PVP-free polycarbonate filters are a type of laboratory equipment used for filtration purposes. These filters are made of polycarbonate material and do not contain polyvinylpyrrolidone (PVP). They are designed to separate particles or molecules from a liquid or gas sample.
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Lab products found in correlation
3 protocols using pvp free polycarbonate filters
1
Boyden Chamber Chemotaxis Assay
2
Neutrophil Functional Assays and DMR Measurements
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All chemicals and reagents were purchased from Sigma–Aldrich, unless specified otherwise. Assay buffer for functional neutrophil assays was made from Dulbecco`s modified PBS (with 0.9 mmol/L Ca2+ and 0.5 mmol/L Mg2+; 0.1% BSA, 10 mmol/L HEPES, and 10 mmol/L glucose, pH 7.4). HBSS buffer for DMR measurements was from ThermoFisher. Anti‐CD16‐PE‐Cy5(3G8), anti‐CD63‐FITC(H5C6) and anti‐CD11b‐PE(ICRF44) were from Becton Dickinson (Vienna, Austria). CellFix and FACS‐Flow were from Becton Dickinson (Vienna, Austria). PVP‐free polycarbonate filters were from NeuroProbe (Gaithersburg, USA). Fluo‐3‐AM was supplied from Life Sciences (Vienna, Austria). Fixative solution was prepared by adding 9 ml of distilled water and 30 ml of FACS‐Flow to 1 ml of CellFix. DMR experiments were performed using the Epic® System (Corning, USA) in fibronectin‐coated cell‐based biosensor plates (Corning, USA). FR900359 was a kind gift of Evi Kostenis and Gabriele König (University of Bonn, Germany).
3
Boyden Chamber Assay for U2OS Cell Invasion
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U2OS cells invasiveness was evaluated using Boyden chamber assay. The method is based on the passage of the cells across 8 µm pore size polyvinyl pyrrolidone (PVP)-free polycarbonate filters (Neuro Probe, Inc., USA) precoated with Matrigel (50 μg/filter), placed between the two wells of the Boyden chamber. CM from Doxo-treated fibroblasts (SM), Quercetin pre-treated Doxo-treated fibroblasts (QSM), Quercetin pre-treated fibroblasts (QM), and control cells (CM) were placed in the bottom wells of the chamber. U2OS cells resuspended in complete DMEM High supplemented with 2 % FBS were seeded in the upper chamber (2 × 10 4 cells/well) and incubated overnight. The filters were removed and fixed in Methanol for 1 h. Non-migrated cells attached to the upper surface of the filter were scraped, while migrated cells, adherent on the lower filter surface, were stained with Crystal violet 0.01 % and counted using a light microscope (40 × magnification). Each point was performed in triplicate, and mean values of migrated cells for each point were calculated and expressed in relation to control.
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