Transwells with 8 μm pores

DNT (0.4 × 10⁶) from ConA-treated C57BL/6 GFP⁺ mice were placed in the top chamber of Transwells with 8-μm pores (Corning Inc., Acton, MA, USA). WT C57BL/6 splenocytes (2 × 10⁶/ml) stimulated by ConA (5 μg/ml) were placed in the lower wells. The lower chambers contained complete medium with 0, 2.5 or 5 μg/ml CXCR3-173 (a neutralizing mAb against mouse CXCR3, BioXCell, West Lebanon, NH, USA). DNT cultured alone in the upper chamber served as negative controls. Transwell plates were incubated at 37 °C with 5% CO₂ for 6 h, after which, the upper chambers were removed. Then, GFP⁺ DNT that had migrated to the lower wells were counted. Migration was quantified using inverted fluorescence microscopy by analyzing at least 10 random fields from each of three replicate filters for each experimental condition.

Cells were seeded in six-well plates (1.0 × 10² cells/well) and cultured in M10 medium for 15 days. For migration and invasion assays 5 × 10⁴ cells/chamber suspended in MEM were added to the upper compartments of the Transwells with 8 μm pores (Corning Incorporated, Corning, NY, USA). M10 was added to the wells for chemotactic attraction, followed by incubation of the plates for 12 h for migration and 36 h for invasion assay. Cell invasion was assessed by their ability to transpose both a Matrigel^® coat (BD Biosciences, San Jose, CA, USA) and barrier (Corning Incorporated, USA). Colonies, migrating and invading cells were fixed with 70% ethanol solution (Merck Millipore, Darmstadt, Germany), stained with 0.5% crystal violet (ThermoFisher, Waltham, MA, USA) in 10% ethanol solution and counted. For anchorage independent growth assay, 2.5 × 10² cells were suspended in 500 μL of M10 medium with 0.6% agarose and seeded in 24-well plates coated with the 1% agarose, and covered with M10 medium. After 30 days 50 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 5mg/mL) was added for staining and counting of colonies.

Transwells with 8 μm pores (Corning, Corning, USA) were precoated with Matrigel (1:7 in DMEM, Corning, Corning, USA). Cells were seeded at 5×10⁴/well in serum-free medium in the upper chambers after overnight starvation. The cells were then allowed to invade the lower chamber containing medium supplemented with 10% FBS. The inserts were then fixed with 4% paraformaldehyde for 15 min and stained with 0.5% crystal violet for 15 min. Migrated cells from five random fields were photographed by an inverted microscope and manually counted.