Corning transwells polyester membranes (6.5 mM, 3.0 μM pore size, Sigma Aldrich, CLS3472) were coated with human fibronectin at 10 μg/mL (Sigma Aldrich, F089S) for 1 h at 37 °C. Wells were then washed 2× with sterile PBS and dried overnight. For invasion/migration assays, the bottom chamber contained 600 EBM2 media μL with or without 100 nM N-formyl-met-leu-phe (Sigma Aldrich, 47729-10mg-f) as a chemoattractant. Neutrophils (2.5 × 104) suspended in 200 µL of EBM2 media were placed in the upper chamber and were allowed to migrate for 2 h at 37 °C. Membranes were carefully aspirated, washed twice with PBS and stained with crystal violet for 10 min and rinsed with water until clear. Five images of each transwell were obtained on a Keyence BZ-9000 microscope at ×40 and the area of crystal violet was analyzed using the Keyence Bioanalyzer software. The results from 5 images were averaged together and a percent change in migration was calculated by dividing the percent area of transwell stimulated with a chemoattractant divided by percent area without chemoattractant for each individual patient.
Cls3472
Manufactured by Merck Group
Sourced in United States
CLS3472 is a lab equipment product manufactured by Merck Group. It is a multi-purpose device designed for use in various laboratory settings. The core function of CLS3472 is to facilitate precise and efficient measurements and data collection processes.
Automatically generated - may contain errors
4 protocols using cls3472
1
Neutrophil Migration Assay Using Transwell
2
Chemotaxis Assay for CD14-depleted PBMCs
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The assay for chemotaxis was performed in 24-well plates (Costar, Cambridge, MA) carrying 6.5 mm Trans-well with 3.0 μm pore polyester membrane insert (CLS3472, Sigma). CD14-depleted PBMC were washed once and suspended at 20*106 cells/ml in serum free RPMI 1640 medium. Supernatant were placed in the lower compartment, and cells were loaded onto the inserts at 2*106/100μL each individual assay. Chambers were incubated for 4h in a 5% CO2-humidified incubator at 37°C. After the incubation period, numbers of CD14-depleted PBMC migrating to the lower chamber were counted by flow cytometry (BD Accuri™ C6) using counting Beads (C36950, Thermofisher) and analyzed using FlowJo v10.0.8 (Treestar, Olten, Switzerland). All conditions were tested in triplicate. Statistic evaluation was performed using the Student t test.
3
Evaluating Larval Growth in 3D Skin Models
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First, to ensure that the components within the skin model media did not have any detrimental effects on L4 development, experiments were carried out in which the four experimental co-culture media 3D-1 to 3D-4 were compared. The L4 larvae were plated in duplicates [about 10 worms per transwell (CLS3472; Sigma, USA)] in a 24-well-plate containing 5x103 HUVEC/well and divided into 5 experimental groups. The first four groups were cultured each in one of the four media: 3D-1, 3D-2, 3D-3 or 3D-4 (described above and in S1 Table ). The fifth group, “2D-control” group, was cultured in L4 medium, the established 2-D culture condition for the long-term culturing of L4 [15 (link)]. The worms were observed for motility and growth over a period of a week. A Nikon DS-Fi12 camera and Nikon Eclipse TS100 inverted microscope was used to measure the length of the worms using the NIS Elements version 4.3 Windows based imaging program.
4
Cell Migration Assay with Transwell Inserts
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Cell migration assays were carried out using transwell inserts with pore sizes of 3 μm (polyester membrane, Sigma-Aldrich, CLS3472) or 8 μm (polycarbonate membrane, Sigma-Aldrich, CLS3422), in 24 well plates, as described by Justus et al. [85 ].
Cells were seeded in the top chamber at a density of 1 × 106 cells/mL and incubated for 4 h, at 37 °C and 5% CO2 in a humidified incubator, to allow cell adhesion and spreading. After attachment, cells were supplemented with new culture media, with or without treatment. Culture media with 10% heat inactivated foetal bovine serum (FBS, Invitrogen, 10,099,141) was added to the bottom well to create a chemotactic gradient. Cells were incubated in a humidified incubator at 37 °C and 5% CO2. The chemotactic gradient was renewed every 24 h via addition of new media in both wells. To assess untreated WK1 cell migration over a period of 72 h, separate transwells were run in parallel and fixed at 24, 48 and 72 h, respectively.
The migration of cells, treated with culture media containing 0.1 mg/mL Tf@pSiNPs, 50 μM NFA (Sigma Aldrich, N0630), or 2 mM orthosilicic acid [33 (link)] was characterised using the cell migration assay described.
Cells were seeded in the top chamber at a density of 1 × 106 cells/mL and incubated for 4 h, at 37 °C and 5% CO2 in a humidified incubator, to allow cell adhesion and spreading. After attachment, cells were supplemented with new culture media, with or without treatment. Culture media with 10% heat inactivated foetal bovine serum (FBS, Invitrogen, 10,099,141) was added to the bottom well to create a chemotactic gradient. Cells were incubated in a humidified incubator at 37 °C and 5% CO2. The chemotactic gradient was renewed every 24 h via addition of new media in both wells. To assess untreated WK1 cell migration over a period of 72 h, separate transwells were run in parallel and fixed at 24, 48 and 72 h, respectively.
The migration of cells, treated with culture media containing 0.1 mg/mL Tf@pSiNPs, 50 μM NFA (Sigma Aldrich, N0630), or 2 mM orthosilicic acid [33 (link)] was characterised using the cell migration assay described.
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