Hts transwell 96 well plate

The migration assay was performed in HTS Transwell^®-96-well plates (Corning Incorporated, Corning, NY, USA) with 5.0 µm pore size polycarbonate membranes following the manufacturer’s instructions. BaF3 cells were seeded in the Transwell upper insert and the lower insert contained media with 200 ng/ml of CXCL12 (R&D Systems, 350-NS-010), and were incubated for 6 h at 37 °C and 5% CO₂ after which the cell number in the bottom chamber was counted by Accuri C6 Flow Cytometer (BD Biosciences, Franklin Lakes, NJ, USA). The migration of BaF3 clones was normalized to the values obtained from BaF3 parental cells and the statistical analysis (Student’s t test) was done in GraphPad Prism.

The chemotaxis assay was conducted in HTS Transwell-96 well plates (Corning Incorporated) with 5.0 μm pore size polycarbonate membranes following the manufacturer’s instructions.
Total number of 0.3 × 10⁶ MEC1 cells were seeded in the upper well of the transwell plate. Chemokine gradient was created by addition of CCL19 chemokine (R&D Systems, CCL19/MIP-3beta, 361-MI) in concentration of 100 ng/mL for D4476 dose response testing and 200 ng/mL in case of VANGL2-Venus or VANGL2 siRNA testing to the lower well of the plate. Sterile PBS/0.1% BSA solution in corresponding amount was used in control conditions. Cells were incubated for 6 hours and then number of migrated cells was analyzed by Accuri C6 Flow Cytometer (BD Biosciences). The assessment of cell viability was performed by TMRE staining (Tetramethylrhodamine ethyl ester perchlorate, Sigma-Aldrich) as described previously [10 (link)]. The migration index was calculated as the number of cells (treated or untreated) migrating in response to the chemokine divided by the number of cells migrating toward the control medium only. Graphs show either migration index or number of migrated cells in 20 μL of sample taken from the lower well of the transwell plate.

VSMCs migrations were evaluated by wound healing assay and transwell assay. VSMCs were grown and converged in a 6-well plate. A transverse scratch wound on each monolayer of VSMC was made by using a sterilized 200 µl-tip. The scratched VSMCs were then stimulated with or without resistin (40 ng/ml) in the presence or absence of Rb1 (20 µM) for an additional 6, 18, and 24 h, then the transverse scratch wounds were reexamined for cell migration. Pictures were captured with a phase-contrast microscope, and cell migration was quantified using ImageJ software, which was calculated as the percent of the wound closure area relative to that at the start point (t = 0).
Transwell assay was performed in Transwell chambers (HTS Transwell-96 Well Plate, 5 µm pore size; Corning Inc., USA). Briefly, 1 × 10⁵/ml cells were seeded on the Transwell Inserts in 75 µl of serum-free medium with or without Rb1 (20 µM). The bottom plate contained 235 µl of complete growth medium with resistin at 40 ng/ml. Cells migrated at 37°C for 24 h. After 24 h of culture, the non-migrated cells on the membrane top surface were removed with a cotton swab. After that, the migrated cells on the bottom side of the membrane were stained with 0.1% crystal violet and then destained with PBS.