Millicell insert

Embryonic mouse brains were electroporated at E14.5 and sacrificed 2 days later. After checking green fluorescence under microscope, the embryonic brain was sectioned into 300 μm in artificial cerebrospinal fluid. The cortical slices were transfered onto Millicell inserts (Millipore) in Neurobasal medium (Invitrogen) containing 2% B-27 supplement, 2 mM L-glutamine, and 1% penicillin/streptomycin. The inserts were placed into a glass-bottomed dish for microscope observation.

Chemotaxis towards TGFβ1 was analysed using a modified Boyden chamber assay (24 wells Millicell inserts, 0.8 μm, Millipore). 10,000 cells were seeded within the inserts in a 24-well plate in DMEM without serum. After 4 hours, the inserts were transferred to a new plate containing serum free growth medium supplemented with 0.01 pg/ml to 10 ng/ml TGFβ1 (recombinant human, R&D systems). Cell migration was allowed for 18 hours and cells were fixed with 10% formalin and stained with haematoxylin. After extensive rinsing, the inside of the inserts were cleaned using cotton buds and the membranes were cut and mounted with DPX mounting medium. Migrated cells were quantified by bright field microscopy in at least 8 different fields of view at 20x and normalised to the number of cells migrated without TGFβ1 supplementation (chemotactic index).

Ker-CT cells or HaCaT cells (3 × 10⁵) were resuspended in CnT-PR keratinocyte medium (Cell-n-Tech) and seeded onto MilliCell inserts (MilliPore). Inserts were immersed in growth medium in 60 mm culture dishes. After 3 days, the medium was substituted with CnT-3D Barrier medium (Cell-n-Tech) overnight (16 h). The day after, the medium was removed, and the inserts were put in the low level CnT-3D Barrier medium to induce air-lifting. The Barrier medium was changed every two days for three weeks. Inserts with epidermal organotypic cultures were fixed in 10% formalin buffered solution for 24 h and then processed and embedded in paraffin.

The cells were harvested through trypsinization, then counted and resuspended in RPMI-1640 at a concentration of 1×10⁵/ml. Next, 0.5-ml aliquots of cell suspension were added to the upper chamber of Millicell^® Inserts (Millipore Corporation, Billerica, MA, USA). The upper and lower chambers were separated using a 12-mm pore polycarbonate membrane that was coated with Matrigel™ (Becton Dickinson). Subsequent to 24 h of incubation at 37°C, the remaining cells on the upper side of the chamber were removed using a cotton swab. The cells that had migrated through the pores to the bottom side of the membrane were fixed using 3.7% paraformaldehyde and stained with hematoxylin and eosin. The number of migrated cells was counted in 10 randomly selected fields using a microscope.

Cells were cultured to 70–80% confluence and starved for 18–20 h in serum-free LG DMEM (Life Technologies, Tastrup, Denmark). Transwell migration assay was performed using 12 mm Millicell^® inserts (Millipore). Briefly, adding 5% FBS MEM or specific conditional medium in the lower chamber of well, test cells were plated in the upper chamber in 0.2% FBS DMEM medium and incubated at 37 °C for 16 h. The cells on the surface of the upper chamber were removed by cotton swabs; the membranes in the inserts were removed and fixed by formalin buffer (Sigma-Aldrich) for 5 min, stained in Hemacolor staining solution (Merck, Germany) for 15 min. Transfer the membranes to glass slide (Menzelgläzer, Germany) and scan the whole membrane by a Lecica DM4500 microscope (Olympus, UK). The numbers of migrated cells or sizes of membrane area covered by the cells were determined by Surveyor Turboscan Mosaic acquisition imaging system (Objective Imaging, Cambridge, UK).

A sterile working field was set up for the sectioning procedure. This is to avoid any contamination of the tissue being processed for the sectioning. All tools required for this procedure were sterilized following clinical protocols and placed within reach of the experimenter to prevent delays. The sections were prepared using a vibratome (VT1200; Leica Biosystems). The sectioning chamber was filled with ice-cold preparation medium containing Hibernate-A Medium (Lot No. 1994548; Gibco) supplemented with 13 mM d-glucose (Lot No. SLBX3638; Sigma-Aldrich), 30 mM N-methyl-D-GLucamin (M2004; Sigma-Aldrich), and 1 mM GlutaMAX (Lot No. 1978435; Gibco), saturated with carbogen (95% O₂ and 5% CO₂) for 10 min before the sectioning of resected tissue. Millicell inserts (No. PICM03050; Millipore) were placed in each well of a six-well culture dish with 1 ml of growth medium containing Neurobasal l-Glutamine (Lot No. 1984948; Gibco) supplemented with 2% serum-free B-27 (Lot No. 175040001; Gibco), 2% Anti-Anti (Lot No. 15240-062; Gibco), 13 mM d-glucose (Lot No. RNBG7039; Sigma-Aldrich), 1 mM MgSO₄ (M3409; Sigma-Aldrich), 15 mM Hepes (H0887; Sigma-Aldrich), and 2 mM GlutaMAX (Lot No. 1978435; Gibco).