Quantification of the overall lung caspase-3 activity in lung homogenates and caspase-3 immunohistochemistry on lung sections was performed as described previously (Bem et al. 2010c (link)). To quantify lung epithelial cell apoptosis by immunohistochemistry, the number of cleaved caspase-3-positive epithelial cells (airway and alveolar) was counted in four random high-power fields per lung tissue section.
To obtain single lung cell suspensions for FACS analysis, lungs were perfused with 20 mL PBS through the right ventricle, minced using iridectomy scissors, and digested with collagenase III. Finally, the cells were passed through a 70-μm cell strainer and subjected to RBC lysis, and kept on ice until labeling. The isolated cells from the lung and BALF were Fc-blocked with antimurine CD16/CD32 and labeled with: Annexin V-APC (A35110; Life-Technologies-Invitrogen, Bleiswijk, the Netherlands), LIVE/DEAD fixable far red dead cell stain kit (Life-Technologies-Invitrogen, Bleiswijk and the Netherlands) and the neutrophil marker Ly-G6 (GR1-PE; eBioscience, Hatfield, UK). Annexin V-positive and LIVE/DEAD far red stain-negative cells were considered apoptotic.
To obtain single lung cell suspensions for FACS analysis, lungs were perfused with 20 mL PBS through the right ventricle, minced using iridectomy scissors, and digested with collagenase III. Finally, the cells were passed through a 70-μm cell strainer and subjected to RBC lysis, and kept on ice until labeling. The isolated cells from the lung and BALF were Fc-blocked with antimurine CD16/CD32 and labeled with: Annexin V-APC (A35110; Life-Technologies-Invitrogen, Bleiswijk, the Netherlands), LIVE/DEAD fixable far red dead cell stain kit (Life-Technologies-Invitrogen, Bleiswijk and the Netherlands) and the neutrophil marker Ly-G6 (GR1-PE; eBioscience, Hatfield, UK). Annexin V-positive and LIVE/DEAD far red stain-negative cells were considered apoptotic.
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