Oregon green 488 conjugated gelatin

Glass coverslips (10 mm) were placed in a 24-well plate and precoated with 0.01% Poly-L-lysine (Sigma–Aldrich). The coverslips were then coated with 0.1% Oregon green 488-conjugated gelatin (Invitrogen), crosslinked with 0.5% glutaraldehyde, and coated with 2 μg/ml SDF-1α (PeproTech—RKP48061). Lymphocytes were incubated overnight over the gelatin-coated coverslips. Following incubation, cells were fixed and counterstained with phalloidin 594. Images were acquired with the Zeiss Confocal Laser Scanning Microscope (LSM 510 META) under a ×63 objective lens. The images were analyzed using ImageJ software, and the percentage of degradation was determined for each cell according to the equation: %Degradation area = (Area of degraded gelatin/Area of corresponding cells) × 100.

ECM-degradation by cells on coverslips precoated with Oregon Green 488-conjugated-gelatin (Invitrogen) was analyzed for 18 h, as described in a previous report [22] (link).

Glass-bottomed dishes were treated with 0.1 mg/ml Poly L-lysine (Sigma-Aldrich) and crosslinked with 0.5% glutaraldehyde (Mecoxlane). Then, the dishes were coated with fluorescent gelatin [1:5 dilution of Oregon Green 488 conjugated gelatin (G13186, Invitrogen) with 0.1% unconjugated gelatin (G1393, Sigma)] at 37 ℃ for 1 h. The dishes were then incubated with 5mg/ml sodium borohydride for 15 min, and then incubated with 70% ethanol for 20 min. Three washes with 1×PBS were performed between each step. DMEM media was added to the dishes at 37 ℃ for 1 h before cell plating. After incubating the cells at 37 ℃ for a further 48 h, the cells were fixed in 4% paraformaldehyde (PFA) and permeabilized with 0.5% Triton X-100, rinsed with PBS and blocked in 3% bovine serum albumin (BAS) in PBS. The cells were then incubated with primary antibodies against Tks5/FISH (sc-30122, Santa Cruz) and F-actin/Phalloidin (A12380, Invitrogen), and then incubated with Alexa Fluor 647-labelled secondary antibodies (A-21245, Invitrogen). The samples were observed under a Nikon A1 confocal microscope. Quantification of the degraded gelatin area was performed using Image J software (National Institutes of Health).

Coverslips were prepared with Oregon green⁴⁸⁸ conjugated gelatin (Invitrogen, Burlington, ON, Canada) at a final concentration of 0.5%, as previously described [7 (link)]. Thirty thousand cells were seeded onto each coverslip, incubated for 48 h, and fixed with 4% paraformaldehyde. Nuclei were stained with DAPI, and F-actin was stained with Texas Red phalloidin. Stained cells were visualized using a Zeiss Axioskop fluorescence microscope, and invadosomes were identified by F-actin-enriched areas of matrix degradation. Three fields of 100 cells (magnification 40×) were counted per coverslip to quantify the percentage of invadosome-forming cells.

Coverslips were prepared as previously described [50 (link)], using Oregon-Green⁴⁸⁸-conjugated gelatin (Invitrogen, Burlington, ON, Canada). Forty thousand cells were seeded on each coverslip and allowed to adhere. Following various incubation times as described within the figure legends, cells were fixed with 2% paraformaldehyde for 10 min at room temperature. Nuclei were stained with DAPI and F-actin was stained using Texas-Red-conjugated phalloidin. Cells were visualized and imaged by fluorescence microscopy using an Axioskop 2 phase-contrast/epifluorescence microscope (Carl Zeiss, Inc., Thornwood, NY, USA). Cells forming ECM-degrading invadopodia were identified based on cells with at least one F-actin-enriched area of matrix degradation (characterized by loss of green fluorescence). Three fields of 100 cells (magnification 40×) were counted per coverslip to quantify the percentage of cells forming ECM-degrading invadopodia.