Boyden chamber

Boyden chambers (3422, Costar, USA) were used to perform transwell assay. 3 × 10⁴ CPCs were seeded in the upper chamber in DMEM/F12 medium, while the lower well was filled with DMEM+/F12 medium. The plates were incubated at 37 °C for 24 h. The cells on the upper surface of the upper chamber were gently wiped. Then, the cells migrated to the lower surface of the upper chamber were fixed in 4% paraformaldehyde and stained with crystal violet staining solution (KGA229, Keygen Biotech, China). These experiments were repeated three times.

Migration assay was performed using 24-well transwell migration Boyden chambers (8 μm pore size; Costar, Cambridge, MA, USA) precoated with matrigel (dilution 1:2 in DMEM; BD Biosciences, Bedford, MA, USA). 5X 10⁴ cells were seeded in each filter and serum starved for 4 h at 37 °C. To induce chemotaxis: FGF2 25 ng/ml was added to the lower chamber. After 48 h, cells on the upper side of membranes were removed, while cells migrated on the bottom side were fixed in methanol and stained with toluidine blue. Quantitative analysis was assessed counting for each sample the migrated cells in 10 microscopic fields (objective used: 20X) from three independent experiments. Results have been expressed as mean values ± SD. p values were calculated using Student’s t test and significance level has been defined as p > 0.05.

The invasion of A549 and H1650 cells was measured using Boyden Chamber assays, as described before [18 (link)]. Both the cell lines were subjected to transient transfection using ID1 siRNA, STMN3 siRNA and GSPT1 siRNA. After 24 hours of transfection, cells were rendered quiescent by serum starvation, and then treated with 1 μM nicotine or 100 ng/ml EGF for 24 hours. Briefly, the upper surfaces of the transwell filters were precoated with collagen (100 μg/filter). Matrigel was applied to the upper surface of the filters (50 μg/filter) and dried in a hood. These filters were placed in Boyden chambers (Costar, USA). Following treatment with nicotine and EGF for 24 hours, cells were trypsinized and 20,000 cells were plated in the upper chamber of the filter in media containing 0.1% bovine serum albumin (Sigma, USA) and Nicotine or EGF. Media containing 20% fetal bovine serum was placed in the lower well as an attractant and the chambers were incubated at 37°C for 18 hours. The filters were processed first by fixing them in methanol followed by staining them with hematoxylin. The cells migrating on the other side of the filters were quantitated by counting six different fields in three independent experiments under 20X magnification.

TNBC cells, treated with vehicle or DHEA 10 µM with or without C-C motif chemokine ligand 5 (CCL5) recombinant protein (rh) 20 ng/mL for 24 h, were placed in the top compartments of Boyden chambers (8-μM membranes, Corning Costar, Corning, NY, USA) while a complete medium was added to the bottom within 24 h. Transmigration was quantified as previously described [23 (link)].

Migration assays were carried out in Boyden chambers using filters (8-μm pore size, Costar). Assay medium (DMEM AQ medium supplemented with 20% FBS) was added to the lower compartment, and SW480 and SW620 colon cancer cells, treated as indicated, were added on top of the insert. After incubation for 24 hr at 37°C, filters were fixed. Non-migrated cells were scraped off the upper side of the filters and the filters were stained with crystal violet. Images were taken under a phase-contrast microscope. The number of migrated cells was quantified after Crystal Violet elution with 30% (v/v) acetic acid in distilled H₂O and 100 μl transferred to a 96-well plate. Absorbance was read at 595 nM.

Cell migration assays were performed in 24-well transwell Boyden chambers (8 μm pore size; Costar, MA, USA) according to the vendor's instructions. Briefly, 100 μL cell suspension (without serum) containing 5×10⁵ cells/mL was seeded into the upper migration chamber. A 600 μl medium containing 10% FBS which served as the chemo-attractant was added to the lower chambers of the transwell plates. After incubation in 37°C for 20 h, the media in the lower chamber were discarded. A 600 μL 1% glutaraldehyde was added to the lower chambers to fix the cells on the membrane for 15 min at room temperature. The upper chambers were removed and wiped with a cotton swab to clear the residual cells on the upper side. Cells migrating to the lower surface of the polycarbonate membrane were stained with 0.1% crystal violet for 25 min. The membranes were rinsed with Dulbecco's phosphate-buffered saline (DPBS) buffer and photographed by using a microscope (Olympus, DP50, Tokyo, Japan). The areas of cell migration were quantified by counting 3 randomly selected microscopic fields and the digital pixel densitometry of images was analyzed using Image-J software (National Institutes of Health, USA).