35mm glass bottom μ dishes

Type I collagen gels were prepared for imaging of collagen architecture and spheroid invasion assays. Collagen gels of 2.0 mg/mL and 3.0 mg/mL were prepared by diluting the stock collagen solution (8-11 mg/mL) with 10× concentrated DMEM, NaHCO₃, 1N NaOH, milliQ H₂O and neutralized to pH 7.2. To visualize collagen fibers by CFM, fluorogenic DQ-collagen I was mixed with unlabeled diluted collagen as previously described (81 (link)), obtaining a final concentration of 20 µg/mL. Collagen dilutions were performed and maintained on ice until use. Droplets (25 µL) of diluted collagens were spotted in 35mm-glass bottom μ-dishes (Ibidi) and polymerized during 45 min at 19°C, followed by a 30 min incubation at 37°C. The polymerization temperature of 19°C was chosen to provide a matrix architecture comparable to in vivo with a network consisting of a mixture of a few thin bundles and many thick bundles (82 (link)). After completion of collagen polymerization, 1 mL of preheated DMEM supplemented with 5% FBS was added to the dishes. Image acquisition was performed after 24 h of incubation at 37°C with 5% CO₂.

One day before the measurement, HeLa cells were grown at 37°C and 5% CO₂ in DMEM medium (Gibco/Thermo Fisher Scientific, Waltham, Massachusetts, USA) including 10% charcoal-stripped FBS (Gibco/Thermo Fisher Scientific, Waltham, Massachusetts, USA), 1% sodium pyruvate (Gibco/Thermo Fisher Scientific, Waltham, Massachusetts, USA), 1% non-essential amino acids (Gibco/Thermo Fisher Scientific, Waltham, Massachusetts, USA) and 1% GlutaMAX (Gibco/Thermo Fisher Scientific, Waltham, Massachusetts, USA) in 35 mm glass bottom μ-dishes (Ibidi, Gräfelfing, Germany). Before the measurement, we added TAMRA-Fα27 (Pepscan, RC Lelystad, The Netherlands) in a final concentration of 0.27 µM in OptiMEM (Gibco/Thermo Fisher Scientific, Waltham, Massachusetts, USA) and incubated the cells for 4 h at 37°C and 5% CO₂. Afterwards, we imaged the cells on a custom built spinning disk microscope (36 (link)) using a 532 nm laser to visualize TAMRA-Fα27.
For single molecule imaging of TAMRA-Fα27, we followed the same procedure, but grew cells on Delta-T glass bottom dishes (Bioptechs Inc., Butler, Pennsylvania, USA). The final concentration of TAMRA-Fα27 was 2.7 nM. We imaged single TAMRA-Fα27 molecules using HILO microscopy (36 (link)) with a 561 nm laser at 1 kW/cm² and 10 ms acquisition time.