Transwell chamber

Transwell chambers (Corning, NY, USA) were applied for the measurement of cell migration and invasion of NSCLC cells in this study. Transwell chambers precoated with Matrigel (Corning, NY, USA) were used for invasion assay, while the Transwell chambers without Matrigel coating were used for migration assay. The transfected cells with a density of 2 × 10⁵ cells/well, seeded into a chamber. The upper chambers with serum-free medium and the low chambers were filled with culture medium supplemented with 10% FBS as a chemoattractant. The cells in the lower chambers were stained after 48 h of incubation and observed for counting under an inverted microscope (Olympus Corporation, Tokyo, Japan).

Cell migration and invasion assays were performed as previously described (20 (link)). Transwell chambers (Corning, Corning, NY, USA) equipped with 8-μm pore insets were used for the migration and invasion assays. For the migration assay, Transwell chambers (Corning, Corning, NY, USA) containing 8-μm pores were uncoated with Matrigel in the upper chamber. For the invasion assay, Transwell chambers (Corning, Corning, NY, USA) containing 8-μm pores were coated with 100 μl of 1:8-diluted Matrigel (BD Biosciences) in the upper chamber. Briefly, 100 μl of cell suspension (8 × 10⁴ cells) of serum-free medium was plated in the upper chamber; medium with 10% FBS was added to the lower chamber. After incubating 18 h for migration and 22 h for invasion, we removed the cells of the upper chamber and then stained the migration and invasive cells of the lower chamber using crystal violet solution. The results were photographed by light microscopy and counted by ImageJ. The experiment was done in triplicate.

The in vitro DC chemotaxis assay was carried out in pore polycarbonate filters of 24-well Transwell chambers (Corning Costar, New York, USA), as previously reported.29 (link) Briefly, the top chamber was covered with 5×10⁴ DCs in 200 µL of BSA medium (0.5%), and the lower chamber was filled with the indicated percentages of supernatants from NDV-MIP3α-treated, NDV-WT-treated, or PBS-treated tumor cells to a volume of 750 µL. The Transwell chambers were incubated at 37°C for 3 hours in a 5% CO₂ atmosphere. Thereafter, the filter between the top and lower chamber was washed by Hank's balanced salt solution (HBSS), fixed, and stained on a slide. The number of chemotactic DCs on the lower chamber was recorded by a microscope system (MT20, OSIS, Germany) at 200× magnification. The results of five high-power fields were quantified.

The in vitro cell migration assay was performed using transwell chambers (8 μm pore size; Costar), and transwell invasion assay was conducted using the transwell chambers (8 μm pore size; Costar) and Matrigel (BD Biosciences, San Jose, CA, United States), according to the manufacturer’s instructions. For migration and invasion assays, media containing 20% FBS in the lower chamber served as a chemoattractant. After 24 or 48 h, the nonmigrating or noninvasive cells were removed from the upper face of the filters using cotton swabs. The migrated and invaded cells located on the lower side of the chamber were fixed with 4% paraformaldehyde and stained using crystal violet, air dried, photographed and counted. The number of migratory and invasive cells were counted in 5 randomly selected visual fields from the central and peripheral portions of the filter.

The effect of Lube S on the invasion process of MDA-MB-231 cells was evaluated in vitro using the Transwell chambers (Corning Costar, Cambridge, MA, USA). Matrigel (Corning, Bedford, MA, USA), diluted at 0.3 mg/mL in DMEM medium without FCS, was applied onto the 8 mm pore size polycarbonate membrane filters of the Transwell chambers, and incubated at 37 °C for 16 h, allowing its polymerization. MDA-MB-231 cells, at 4 × 10⁴ cells/well and suspended in DMEM medium without FCS, were then seeded on the upper part of the chamber at a final volume of 200 μL. The bottom chamber was filled with 800 μL of DMEM medium containing 10% FCS. After 2 h, cells were treated with Lube S at its IC₃₀, IC₅₀, and IC₇₅ for 16 h.
Cells migrating to the lower chamber through the Matrigel basement were fixed with paraformaldehyde 3.7%, stained with 0.1% crystal violet, and photographed. Invasion of MDA-MB-231 cells was defined in terms of percentage of invasive cells vs. untreated controls after counting three randomly selected fields.

Based on the instructions of manufacturer, Transwell chambers (8 µm pore size; Costar) were employed for the test of cell migration, and Matrigel (BD Biosciences, San Jose, CA, United States) together with Transwell chambers (with a pore size of 8 µm; Costar) were applied for Transwell invasion test. For the analysis of invasion and migration, the medium involving FBS (20%) in lower chamber was applied as a chemical attractant. After 1 day, non-invasive or non-migrating cells were removed from the upper filter surface employing cotton swabs. The invasive and migrating cells situated at the chamber lower side were fixed with paraformaldehyde (4%), which were stained by crystal violet for half an hour. Subsequently, the cells in the upper part of the chamber were wiped with cotton swab, and the cells existing in the chamber bottom were imaged and next counted under randomly five fields with microscope.