Zen 2010b software

Cells were plated onto 35 mm-glass-bottomed dishes (Greiner Bio-One) one day prior to the experiment and incubated on the microscope stage at 37°C in humidified 5% CO₂. Two Carl Zeiss confocal microscopes were used (LSM710, AxioObserver and LSM780 AxioObserver) with Fluar 40x/1.30 NA Oil objectives. The 488 nm (ATOF set at 4%) line from an argon ion laser was used to excite the p65-EGFP fusion protein and emitted light between 490–540 nm was detected through pinholes set to 5μm. Image capture was performed using the Zeiss Zen 2010b software. Quantification of p65-EGFP nuclear fluorescence (or cytoplasmic fluorescence at t0) was performed using Cell Tracker (version 0.6) using region of interest analysis [62 (link)]. The data was exported as mean fluorescence intensity. TNFα-induced nuclear NF-κB responses in U2OS cells were less robust in comparison to previously described single cell oscillations in different cell types [11 (link), 46 (link), 53 (link)]. In particular, the first peak translocation had a relatively low amplitude (Fig 4). A cell was classified to have responded if >10% change in the nuclear NF-κB p65-EGFP level (in comparison to a basal unstimulated level at t = 0) was observed for >6 consecutive imaging time-points (equivalent to >12 mins).

Articular or xiphoid cartilage tissue from p65-DsRed mouse was embedded in the Matrigel matrix (BD Biosciences) in 35-mm glass bottom Cellview dishes (Greiner Bio-one). Images were acquired with a Zeiss LSM 780 Confocal Inverted Microscope in a humidified CO₂ incubator (at 37°C, 5% CO₂) with a C-Apochromat 40×/1.2 W Korr objective. During imaging tissue was treated with IL-1β (5–20 ng/Ml) or TNFα (up to 40 ng/mL). DsRedXP tagged p65 was visualized by excitation with a green helium neon laser (543 nm) and detection through both a 545-nm dichroic mirror and a 560-nm long pass filter. Data capture was performed using ZEN2010B software (Zeiss).