Oregon green 488 conjugate

C6 and RC6 cells were plated in 6-well plates with glass coverslips coated with AlexaFluor488 labeled gelatin (Gelatin from Pig Skin, Oregon Green^® 488 Conjugate, Life Technologies, Waltham, MA, USA). After 24 h, cells were fixed with 4% paraformaldehyde and co-stained with Hoechst 33342 and ActinRed 555. Cells and degradation area were analyzed under a Zeiss Axiovert inverted fluorescent microscope (Carl Zeiss Foundation, Heidenheim, Germany). Volume of the dark area caused by degradation of gelatin was measured in ImageJ software (1.48, Microsoft, Redmond, WA, USA) and normalized in relation to the volume of the cell. At least 100 cells were analyzed per experiment.

Human Alpha Thrombin (HT 1002a) and human plasminogen (HPg 2001) were acquired from the Enzyme Research laboratories (Bulimba, Australia). Human Fibrinogen (Oregon Green^™ 488 Conjugate) and Interleukin-1β were purchased from Invitrogen (Victoria, Australia). Recombinant tissue-type plasminogen activator and d-Dimer (d2d) ELISA Kit were ordered from antibodies-online Inc. (Atlanta, United States).

For ECM degradation assay, glass-bottom dishes were coated with Gelatin From Pig Skin, Oregon Green® 488 Conjugate (Invitrogen) and then treated with 0.5% glutaraldehyde as described earlier [21 (link)–23 (link)]. Cells were cultured on these glass-bottom dishes in DMEM, fixed and stained with anti-cortactin antibody or Rhodamine Phalloidin (Cytoskeleton). Fluorescent images were obtained using a laser scanning confocal imaging system (OLYMPUS FV1000). Cells in which dot-like degradation of Alexa-gelatin was observed were judged as positive for invadopodia.

Collagen type I matrix (Collagen I, Rat Tail - Gibco) was prepared at a 2 mg/ml concentration with 10% PBS phosphate buffer 10X, 0.26% NaOH 1 N and 10% fluorescent gelatin (Gelatin from pigskin, Oregon green 488 conjugate – Invitrogen). Neutrophils (5 × 10⁴) were then added to the matrix and cultured for 3 h at 37°C and 5% CO₂, fixed with PFA 4% for 15 min, and visualized with a Leica DMi8 inverted fluorescence microscope. Quantification of matrix degradation was assessed by measuring the pixels of gelatin-FITC degradation area using FIJI software. Areas corresponding to 30 cells were quantified for each condition in 3 separate experiments.

Gelatin degradation assay was performed as previously described [13 (link)]. In brief, cells in suspension were added to wells containing equilibrated Oregon Green™ 488 conjugate (ThermoFisher Scientific #G13186) gelatin-coated coverslips, either serum-starved combined or not with mβCD for 2 h and then incubated in serum-containing medium for 6–12 h  or serum-starved for 2 h and then incubated in GM6001-supplemented medium for 6–12 h. Cells were fixed in 4% paraformaldehyde for 30 min, permeabilized with 0.1% Triton X-100 in PBS for 15 min, blocked with 5% Normal Goat Serum in PBS for 1 h, all steps at room temperature, immunolabeled with anti-Cortactin (Cell Signaling Technology #3503, 1:200) overnight at 4 °C, and stained with fluorescent secondary antibodies (ThermoFisher Scientific, 1:1000), Alexa Fluor™ 647 Phalloidin (ThermoFisher Scientific #A22287, 1:200) and Hoechst 33258 (Abcam #Ab228550, 1:1000) for 1 h at room temperature in the dark. Coverslips were then mounted with Dako and examined with a Zeiss Cell Observer Spinning Disk (COSD) confocal microscope using a plan-Apochromat 63 × NA 1.4 water immersion objective and the same settings for illumination. Quantification of total gelatin degradation area per total cell area was done using ImageJ/Fiji [13 (link)].