Computational protocol: Lysosomal storage and impaired autophagy lead to inflammasome activation in Gaucher macrophages

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Protocol publication

[…] Primary macrophages were harvested and sonicated at 4 °C in RIPA Lysate buffer (50 mm Tris‐HCl (pH: 7.4), 150 mm NaCl, 1% NP‐40, 0.5% Na‐deoxycholate, 0.1% SDS, and protease inhibitor). After quantification with BCA (Thermo scientific, Rockford, IL, USA), 10 μg of the lysate was separated by Novex® NuPAGE® SDS‐PAGE Gel System (Life technologies) and transferred to iBlot PVDF membranes (Life technologies). Blots were blocked in 1:1 PBS, Odyssey Blocking Buffer (LI‐COR bioscience, Lincoln, NE) for 1 h at RT. The blocked membrane was incubated in blocking buffer containing 0.1% Tween‐20 and the respective primary antibodies LC3A/B (Cell signaling, Danvers, MA, USA, 1/1000), Atg7 (Cell signaling, 1:1000), histone H3 (Cell signaling, 4499, 1:2000), VPS34 (Sigma, V9764, 1:1000), SQSTM1/p62 (Abcam, Cambridge, MA, USA, 1:1000), β‐actin (Abcam 1:1000), ASC (Enzo life science, Farmingdale, NY, USA 1:1000), caspase‐1 (Enzo life science, 1/1000), NFĸB (Origene, Rockville, MD, USA, 1:500), GAPDH (GeneTex, Irvine, CA, USA, 1:2000), Atg16L1 (Bethyl, Montgomery, TX, USA, 1:2000), cathepsin D (R&D systems, 1:1000), IL‐1β/IL‐1F2 (R&D systems, 1:1000)] overnight at 4 °C, followed by three 15‐min washes, and then was incubated in blocking buffer containing 0.1% Tween‐20 (Sigma), 0.01% SDS, and IRDye®, 680RD secondary antibody 1:10 000 (Li‐CORE Bioscience, Lincoln, NE, USA) for 1 h at RT. The blot was imaged using the LI‐COR Odyssey imaging system (Li‐CORE Bioscience, Lincoln, NE, USA) and quantified using imageJ software. [...] Human macrophages were plated on glass chamber slides and, after various treatments, were fixed with 4% paraformaldehyde for 30 min. For LC3 staining, cells were fixed with cold acetone for 10 min. After one‐step washing with PBS, cells were blocked in PBS containing 0.1% saponin, 100 μm glycine, and 2% donkey serum (2 h) followed by incubation with antibodies against LC3B (Sigma), SQSTM1/p62 (Cell signaling), ASC (Enzo Life science), cathepsin D (R&D systems), Lamp2 (Hybridoma, Columbia, MD, USA), p65‐NFkB (Origene) for 2 h. Then, cells were washed with PBS three times for 5 min followed by incubation with secondary antibodies. Cells were mounted with Vectashield plus DAPI (Vector laboratories, Burlingame, CA, USA) and were imaged using a Zeiss 510 META laser scanning microscope (Carl Zeiss microimaging Inc., Germany). Images were acquired using a 60X oil DIC objective. Images also were taken with Z‐stack (0.3–0.8 μ) and were analyzed using imaris software (Zurich, Switzerland) for co‐localization and surface measurement. [...] Statistical analyses were performed using graphpadprism6.0 software (La Jolla, CA, USA). Significance was determined by a Student's t‐test. Data from two groups or >2 independent variables were analyzed by one‐way ANOVA (nonparametric test; Kruskal–Wallis test). Data are presented as mean values ± SD. Significance levels between controls and patient macrophages were set when P < 0.05(*), P < 0.01(**), P < 0.001(***) and P < 0.05(#), P < 0.01(##), P < 0.001(###) between different conditions. […]

Pipeline specifications

Software tools ImageJ, Imaris, GraphPad Prism
Applications Miscellaneous, Laser scanning microscopy, Microscopic phenotype analysis
Organisms Homo sapiens
Diseases Gaucher Disease, Neoplasms, Parkinson Disease, Genetic Diseases, Inborn