Imaging Membrane Dynamics in Macrophages

Cells were plated onto 35 mm-glass bottomed dishes (Greiner Bio-One, Monroe, NC, USA) and incubated on the microscope stage at 37 °C in humidified 5% CO₂. For monitoring membrane permeabilisation, cells were incubated with PI (5 μg/ml). A Zeiss LSM710 (Oberkochen, Alemania) confocal microscope with a Plan-apochromat × 63 1.3 NA oil immersion and × 40 1.3 NA objectives was used to visualise release of IL-1β. Image capture was performed using the ‘Zen 2010b SP1' Zeiss software. Alternatively, macrophages were imaged with a Nikon Eclipse Ti microscope equipped with a 40 × /0.60S Plan Fluor objective and a digital Sight DS-QiMc camera (Nikon, Tokyo, Japan) and the NIS-Elements AR software (Nikon). Time-lapse microscopy images were quantified either with ImageJ (US National Institutes of Health, Bethesda, MD, USA) or Cell Tracker (version 0.6, Pittsburgh, PA, USA).^{42 (link)} To quantify changes in plasma membrane dynamics over the time, macrophages were labelled with CTB-AF647 (1 : 1000 dilution) for 30 min at 37 °C and imaged using the Nikon Eclipse Ti microscope as stated above. Inverted fluorescence images converted to grey scale were used for quantification of the mean grey value as relative fluorescence units (RFUs) in different regions of interest of the plasma membrane of each cell using ImageJ (US National Institutes of Health).