Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology
Article Title: Free actin impairs macrophage bacterial defenses via scavenger receptor MARCO interaction with reversal by plasma gelsolin
Figure Lengend Snippet: Free actin binds human alveolar macrophages in vitro in a scavenger receptor (SR)-dependent fashion. Scanning cytometry fluorescence imaging. Human GM-Mϕs were incubated (37°C, 40 min) with or without the pan-SR inhibitor PolyI (25 μg/ml), washed in Dulbecco’s phosphate-buffered saline (DPBS), and exposed to 40 μg/ml Alexa Fluor 647-conjugated actin from rabbit skeletal muscle, 40 μg/ml Alexa Fluor 647-conjugated albumin from bovine serum albumin (BSA), or vehicle alone (4°C, 30 min). Cells were labeled with Hoechst and Cell Mask Blue. Representative images are collapsed confocal stacked images. A–E : cells incubated with A647-actin ( n = 5 experiments, 15 wells total; A ), A647-actin with PolyI ( n = 2 experiments, 6 wells total; B ), vehicle control ( n = 5 experiments, 15 wells total; C ), A647-BSA ( n = 5 experiments, 15 wells total; D ), or A647-BSA with PolyI ( n = 2 experiments, 6 wells total; E ). F : value of A647 fluorescence for total cell cytoplasm above baseline staining for all cells, showing increased binding of actin by human GM-Mϕs compared with equivalent concentrations of control protein albumin. Actin binding is significantly reduced in the presence of pan-SR inhibitors. Data are mean fluorescence (FL) ± SE; **** P = 0.0001; ●, ■, ▲, ◆, gray circles, and gray squares all correspond to samples from groups defined in the x -axis.
Article Snippet: Remaining cells were washed twice in DPBS, and all cells were exposed to 40 μg/ml A647-actin, 40 μg/ml A647-BSA (Molecular Probes, Thermo Fisher Scientific), or vehicle alone (GAB, 50% RPMI 1640, 0.5% BSA) while protected from light at 4°C for 30 min.
Techniques: In Vitro, Cytometry, Fluorescence, Imaging, Incubation, Labeling, Staining, Binding Assay