Costar transwell cell culture inserts

We assessed cell migration utilizing Costar Transwell cell culture inserts (Corning Incorporated, Corning, NY, USA) following manufacturer’s guidelines. After 1 d incubation, our team erased cells from transwell chamber upper surfaces using cotton swabs. We fixed these cells on the lower surfaces using methanol for 10 min, which we stained with crystal violet. We photographed and calculated the percentage of stained cells in five fields that were selected at random.

Migration of the lens epithelial cells was assessed by transwell assay. Briefly, lens epithelial cells were harvested and pre-incubated with TGF-β2 (2 ng/ml) and the following reagents for 1 h: (i) scFv O52 (50 μg/ml; negative control), (ii) scFv Fn52 (50 μg/ml), (iii) scFv Fn52 RGDS (50 μg/ml), (iv) RGDS peptide (5 μg/ml), (v) scFv Fn52 (50 μg/ml) + RGDS peptide (5 μg/ml) (vi) scFv Fn52 + scFv Fn52RGDS (25 μg/ml each). Cells were seeded on the filter in the top chamber of the trans-well plate (8.0-μm pore; Corning^® Costar^® Transwell^® cell culture inserts) in a 24-well format, in a total volume of 300 μl. The lower chamber contained the chemoattractant (fibronectin, 0.02 μM, purified from human plasma). The plates were incubated for 8 h at 37 °C and 5% CO₂. Migration of the cells was evaluated by staining the cells migrated on the lower surface of the filter using crystal violet stain and OD 560 of the eluate. All experiments were performed four times.

Corning Costar transwell cell culture inserts (8 µm pore size, 24 mm diameter) were coated with 0.5 ml Matrigel (2 mg/ml) that gelled at 37 °C for 2 h. 2.5 × 10⁵ cells were seeded in the upper chamber in RPMI-1640 (BT549) or DMEM/F12 medium (MCF-10A) without fetal bovine serum or growth factors. Complete growth media were added to the lower chamber. After 24 h of incubation in a tissue culture incubator, cells and Matrigel in the upper chamber were removed with a cotton swab. The cells that migrated through Matrigel to the bottom of the transwell were visualized with Giemsa stain and photographed. Cells in 24 fields (6 fields per well using a 20 × objective, quadruplicates) were counted for each group.

Hep3B cells lentivirally transduced with CRISPRa were seeded at the concentration of 5 × 10⁴ in serum-free medium in the inner chamber of Costar^® Transwell cell culture inserts (6.5 mm diameter, 8 μm pore size, Corning, Cat. No. 3422). A complete medium containing 10% FBS was added to the outer chamber as a chemoattractant, and the cells were incubated for 48 h. The inserts were stained as per protocol using a staining solution containing 0.5% crystal violet dissolved in 25% methanol and sterile water for 10 min. Images from 12 fields of view were acquired with an inverted Leica light microscope to quantify the percentage of cells that had migrated, based on biological triplicates, using ImageJ software.

Transwell invasion assays were performed using Costar Transwell cell culture inserts (Corning) with an 8-μm pore size PET membrane in a 24-well plate. The lower chambers of the transwell plates were filled with 500 μL of cell-appropriate culture media. MDA-MB-231 or SUM159 cells were pre-treated with 0.5 or 1 μM of MBZ for 48 h. The cells (20,000/well) were resuspended in 150 μL of medium containing only 1% FBS and placed into the upper well. After 24 h, cells in the upper chamber were removed with a cotton swab, and those that migrated through the pores on the lower surface were fixed in 100% ethanol (EtOH) and stained in 0.2% crystal violet dye. Five random fields of each pore were imaged using a Cytation 5 (BioTek Instruments) equipped with an Olympus–UPLFLN 10XPh phase objective. For MDA-MB-231 cells, the percent area and total area were quantified using ImageJ software using a custom macro with the following steps: Each image was converted to 16-bit, the scale was converted from pixel to μm, the background was subtracted using a rolling ball radius of 10 pixels and light background, and a threshold was set to highlight crystal violet positive areas of the wells. The macro was altered to include the color threshold feature, the “hue” slider was used to highlight the cell area, and images were processed at 8-bit to quantify the % area for SUM159 cells.