Endothelial cells were plated on collagen I rat tail-coated coverslips. When grown in co-culture, astrocytes plated on the top of transwell tissue culture inserts (BD Falcon), were transferred on the endothelial monolayer. Cultures were checked for confluency and then kept for 2 days at 37°C before treatment, as indicated. Endothelial cells were fixed in ice-cold acetone for 15 min and subsequently in ice-cold methanol for 20 min. Anti-VE-cadherin primary antibody (1:100, SantaCruz Biotech, Cat. # sc-52751, Lot #JO914), was incubated in 0.1% Triton X-100 at 4°C overnight. Secondary antibody (Donkey anti-rabbit Alexa Fluor 546, Invitrogen, Cat. # A100040, Lot #1218269) was incubated for 45 min at room temperature. Cells were imaged using an epifluorescent microscope Zeiss Observer.Z1 microscope equipped with the Apotome.2 acquisition system connected to a digital camera.
Transwell tissue culture inserts
Manufactured by BD
Sourced in United States
Transwell tissue culture inserts are a type of laboratory equipment used for studying cell migration, invasion, and permeability. These inserts consist of a porous membrane supported by a permeable support, which allows for the creation of a two-chamber system. This setup enables the investigation of cellular interactions and responses in a controlled environment.
Automatically generated - may contain errors
3 protocols using transwell tissue culture inserts
1
Endothelial-Astrocyte Co-Culture Imaging
2
Transwell Assay for Cell Migration
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The MMT cells were seeded at a density of 10,000 cells per well in 24-well Falcon™Companion plates (Thermo Fisher Scientific). Twenty hours later, the medium culture was refreshed and 15,000 MSS-31 cells per well were seeded under serum-free conditions upon Transwell tissue culture inserts with 8.0 µm pore size (Falcon™), at a density of 25,000 cells per well. Alternately, MSS-31 cells were seeded at a density of 20,000 cells per well in 24-well Companion plates (Falcon™). Twenty hours later, the medium was replaced and MMT cells were seeded in Transwell tissue culture filters with 8.0 µm pore size (Falcon™), at a density of 25,000 cells per well. The cells were allowed to migrate for 30 h, then non-migratory cells were removed from the top of the filter by swiping with humidified cotton swabs. Cells that had migrated through the filter pores to the lower face of the inserts were fixed in 4% paraformaldehyde in PBS, their nuclei were stained with Hoechst at 5 mg/l in PBS, and counted. Values from the control conditions were set as the reference value of 1. Data are expressed as means of 3 independent experiments.
3
Transwell Chemotaxis Assay for Murine Spleen T Cells
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Spleen T lymphocytes (3 × 106 in HBSS without Ca2+/ Mg2+) were placed in the upper chamber of 5.0 μm pore diameter transwell tissue culture inserts (BD Falcon, USA). Transwell inserts were placed in the individual wells of a 24-well cell culture plate containing assay buffer or lung homogenates from naïve, sham-operated and CLP-operated mice, neutralized (30 min, 37 °C) with anti-CCL2 mAb (2.5 ng/well), anti-CCL3 mAb (200 ng/well) or anti-CCL5 mAb (50 ng/well). The recombinant chemokines rmCCL2 (2.5 ng/well), rmCCL3 (4 ng/well) and rmCCL5 (4 ng/well) (R&D Systems, USA) were used as positive controls. After 2 h, the migrated cells were counted, labeled as described above, and analyzed by FACScalibur. Results are expressed as chemotactic index, generated by using the number of cells that migrated towards buffer as comparison.
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