Co2 independent culture medium

Video microscopy measurements were performed and analyzed as described previously [36 (link)]. Briefly, cells were seeded in 24-well plates (Corning Incorporated, USA) and incubated overnight in DMEM medium supplemented with 10% FCS. CO₂-independent culture medium (Gibco-BRL Life Technologies, UK) supplemented with 10% FCS and 4 mM glutamine was applied and the plate was transferred to the custom designed incubator built around an inverted phase-contrast microscope (World Precision Instruments, USA). The experiment was performed at 37 °C in room ambient atmosphere. Images were taken every 10 mins from three neighboring microscopic fields in each well for 72 h. Migration data was captured with a manual cell-tracking program. The parameter migrated distance is calculated by averaging for each cell the displacement for the 48–72 h period after treatment, in at least three microscopic fields.

Mammary glands 2, 3, 4 and 5 were dissected from 8- to 12-week-old female mice, and the lymph nodes were removed before processing. After mechanical dissociation into pieces, the tissue was digested in CO₂-independent culture medium (Gibco) containing 3 mg/ml collagenase A (Roche) and 100 U/ml hyaluronidase (Sigma), supplemented with 5% bovine calf serum for 90 min at 37°C, followed by 0.25% trypsin-EDTA for 1-2 min, 5 mg/ml dispase (Roche) and 0.1 mg/ml DNase (Roche) for 5 min, and 0.64% NH₄Cl for 3 min. Samples were then filtered through a 40 µm mesh and labeled.

Methods of applying EFs have been described previously [5 (link), 25 (link)]. Briefly, cells were seeded into an electrotaxis chamber on a dish (Falcon tissue culture dishes, BD Biosciences, Franklin Lakes, NJ, USA) and left to adhere overnight in a 5% CO₂ incubator. Then a No.1 coverglass was applied as a roof and sealed with high vacuum silicone grease (Dow Corning Corp., Midland, MI, USA) so that the final dimensions of the chamber were 24 mm × 8 mm × 0.2 mm. CO₂ independent culture medium (Gibco) plus 10% FBS was used to maintain stable pH. Direct current was applied through agar-salt bridges connecting silver/silver chloride electrodes in Steinberg's solution to pooled medium on each side of the galvanotaxis chamber. Cells were exposed to 0-200 mV/mm steady EFs for the indicated periods of time. Time-lapse images were acquired using a Live Cell Station (Delta Vision, API, USA).
Stattic was used to attenuate STAT3 activation in H1650-M3 cells. Cells were treated with 0.5μM Stattic for 2 hours, then exposed to an EF of 100 mV/mm for 2 hours in the presence of the inhibitor. To activate STAT3 in Cav-1 KD H1650-M3 cells, IL-6 was added to the culture medium for 6 hours, then cells were subjected to EF stimulation in the continuous presence of IL-6.