Transwell cell culture insert

Migration was analyzed by Boyden chamber assay using Transwell cell culture inserts (8.0 μm pore size; Costar, Cambridge, MA). Adjusted viable cell concentration was counted with trypan blue exclusion. Mouse embryonic fibroblasts were plated (5 × 10⁵ cells/ml in serum-free DMEM) in the upper chamber. The lower chamber containing various growth factors indicated in the figure legend. After 5 h, the cells remaining on the upper surface of the membrane were wiped off, and cells migrating to the lower surface in triplicate wells were visualized with Diff-Quick stain (Sysmex, Japan) and counted in each of four randomly chosen light microscopic fields at × 20 objective.

TEER was measured by impedance spectroscopy using the cellZscope (NanoAnalytics, Münster, Germany). A total of 100 000 HUVEC cells were seeded on Corning Costar Transwell cell culture inserts (5 µm pore size, Costar #3421) and grown for 2 days. For measurements, the basal electrode was overlaid by 500 µL equilibrated medium. After inserting filters, 275 µL of medium were added to the apical surface and the apical electrode was placed into the apical liquid. The measurements were performed immediately after positioning of apical electrodes. The software package provided with the instrument (NanoAnalytics, Münster, Germany) was used for data acquisition and analysis.

Transwell cell-culture inserts (Catalogue: 3450, pore size: 0.4 μm; Corning Costar Corp., NY, USA) were placed in RPMI-1640 medium with 10% FBS and 1% antibiotics in the upper and lower compartments. The heights of the medium in the upper and lower compartments were maintained at similar levels, so the bulk flow was not due to a hydrostatic pressure gradient. PCs were resuspended at 5 ×10⁴/well in the lower chamber (a 6-well plate), which contained 40 mM HG medium; the upper chambers containing the HG medium were seeded with 5 × 10⁴/mL control PTECs or PTECs transfected with Lenti-virus (Lenti-Bim-shRNA) as described in the “Bim Lentiviral vector construction” section below. There was communication between different cells in the system through a Polyester (PET) membrane for 24 h, 48 h, and 72 h. As controls, PCs were also cultured in 6-well plates under normal glucose (NG) and HG medium without inserts.
The experimental groups included: monoculture of PCs in NG and HG, coculture of PCs with control PTECs in HG, coculture of PCs with PTECs transfected with Lenti-Bim-shRNA (PTEC-Bim-shRNA) in HG, and coculture of PCs with PTECs transfected with negative control virus (PTEC-Bim-shNC) in HG. All co-cultures were set up in triplicate.

Transwell cell culture inserts (well size: 0.4 μm; Corning Costar) were placed in RPMI 1640 medium with both upper and lower chambers containing 10% inactivated FBS and 1% double antibodies. In vitro expanded B-cells cultured for 12 days were resuspended at a density of 5×10⁵/well in the lower chamber (6-well plate), which contained both 10 μg/mL of LPS and 50 ng/mL of IL-4. Mouse podocytes (MPC-5) (C2046; WHELAB BIOSCIENCE LIMITED, Shanghai, China) were transferred from 33°C to 37°C for 3 days and resuspended at a density of 5×10⁵/well in the upper chamber, and cells were grown adnexally to 60%–70% at 48 h. Cytokines and metabolites produced by B-cells in the lower chamber were delivered through a polyester membrane. The control group was MPC-5 cultured in normal RPMI 1640 medium (without insert) placed in 6-well plates. The experimental group consisted of co-cultured MPC-5 and rat B-cells from the normal group, rat B-cells from the MN group, rat B-cells from the canagliflozin group, or rat B-cells from the losartan group, respectively.

Transwell cell culture inserts (pore size 8 μm; Corning Co-Star Corp., Cambridge, MA, USA) were placed in RPMI 1640 medium supplemented with 15% foetal bovine serum in the lower compartment. Podocytes with different pretreatments were harvested with trypsin and resuspended in serum-free medium. The upper chambers were seeded with 1 × 10⁵ cells/ml, which were allowed to attach at 37°C for 12 h. Non-migratory cells were then removed from the upper surface of the chambers, and migrated cells on the lower membrane surface were fixed with 4% paraformaldehyde and stained with haematoxylin. The number of migrated cells was counted in 3 separate fields per membrane at 200× using phase-contrast microscopy (Leica Microsystems, GmbH). The data are presented as the mean ± S.D.