Incucyte sx1 live cell analysis system

CASCs were seeded in a 96-well plate at a density of 10,000 cells per well in X-VIVO medium with 2% FCS and 2% P/S. To study apoptosis, a caspase assay was performed using the IncuCyte^® Caspase-3/7 Green Apoptosis Assay Reagent (diluted 1/1000, Sartorius, Schaarbeek, Belgium). Five conditions were added to the medium: 400 µg/mL BSA, 50 µg/mL, 100 µg/mL, 200 µg/mL, and 400 µg/mL AGEs. CASCs cultured in X-VIVO medium without FCS and 2% P/S were used as a positive control. Experiments were performed in triplicate. Images were taken after 24, 48, and 72 h of incubation using the IncuCyte^® S3 Live-Cell Analysis System (Sartorius, Schaarbeek, Belgium). Analysis of the area occupied by apoptotic cells was performed using the IncuCyte^® SX1 Live-Cell Analysis System (Sartorius, Schaarbeek, Belgium). Data were normalized to the positive control (+, X-VIVO medium without FCS).

For performing live-cell fluorescence imaging of infected macrophages, epithelial and endothelial cells IncuCyte® SX1 Live-Cell Analysis System (Sartorius, Germany) was used with green (ex. 440–481 nm/em. 503–544 nm) and red channel (ex.567–607 nm/em. 622–704 nm). Fluorescent signals were monitored in real-time with a microplate reader for 24 h within a tissue culture incubator (37 °C, 5% CO₂) on black OptiPlate 96-well (PerkinElmer, MA, USA) under 20x objective. Cells cytotoxicity (Red object counts) were assessed using DRAQ7 (Invitrogen, Cat. D15105) staining, ASC-specks formation (Green object count) was calculated using IncuCyte SX1 analysis system.

Human embryonic kidney cells HEK293 (ATCC^® CRL1573™), HeLa (ATCC^® CCL-2) and HEK293 TAF1ts [16 (link)] cells were grown at 37 °C in a humidified incubator with 5.6% CO2 in Dulbecco’s modified Eagle’s medium (DMEM; GIBCO, Life Technologies, Thermo Scientific, Waltham, MA, USA) supplemented with 8% fetal bovine serum (GIBCO), 100 units/mL penicillin, and 100 μg/mL streptomycin. The restrictive temperature used for the TAF1ts cells was 39.5 °C. Puromycin was from GoldBio (St. Louis, MO, USA). Light microscopy photographs of cells were performed using an Olympus (Tokyo, Japan) IX70 microscope connected to a DVC camera. Cell colonies expressing YFP or Clover GFP were visualized using the ImageQuant LAS 4000 (GE Healthcare, Piscataway, NJ, USA). The Incucyte^® SX1 live-cell analysis system (Sartorius) was used to photograph cells and quantify cell proliferation and viability.