Triton x solution

Human ASCs were plated at 5 × 10³/cm² and differentiated in chemically defined medium for 28 days. Culture medium and lysed cell supernatant were individually collected on 14 and 28 days. Cells were lysed using 1% Triton-X solution (Sigma-Aldrich, St. Louis, MO). Cell culture lysate was collected by scrapping with a cell scraper (Fisher-Scientific) and briefly sonicated for 10 s using an ultrasonic water bath (Fisher-Scientific) on ice. Total secreted human adiponectin and leptin were measured using ELISA (R&D, Minneapolis, MN) per our prior methods2 (link)10 (link)11 (link)16 (link)17 (link). Absorbance was read using a microplate reader (Biorad, Hercules, CA). DNA content was measured using Quant-iT™ PicoGreen^® dsDNA Assay Kit (Life Technologies) per our prior methods. A high-range standard curve of 1 μg/mL was used for measuring the DNA concentration of the samples per our prior methods. The samples were prepared in a 96 well black bottom plate (Fisher-Scientific) with absorbance read using microplate reader and standard fluorescein wavelengths (excitation ~480 nm, emission ~520 nm; Molecular Devices, Sunnyvale, CA). Total triglycerides and glycerol contents were measured by processing the sample lysate with Free Glycerol Determination kit (Sigma-Aldrich) per our prior methods2 (link)10 (link)11 (link)16 (link)17 (link).

1x10⁵/ml RAW264.7 cells were seeded in 24-well plates (Sarstedt) in pH buffered DMEM (high glucose, HEPES, no phenol red, GIBCO) supplemented with, 1% non-essential amino acids (Sigma) and 10% HI-FBS (GIBCO) and grown at 37°C with 10% CO₂ for 48 h before the infection. Over-night cultures of S. Typhimurium 14028S were centrifuged at 6000 RPM and washed with sterile 1x PBS before being diluted to OD₆₀₀ = 0.4. Bacteria were then opsonized in Balb/cAnCrl mouse serum for 30 min. The old growth medium was removed and fresh pH buffered DMEM without FBS was added before infection at MOI 10. The infection was synchronized by centrifuging the cells for 5 min at 1200 rpm followed by 45 min incubation at 37°C with 10% CO₂. The wells were washed with 1x PBS before adding fresh pH buffered DMEM + gentamicin (100 mg/l). Cells were incubated 45 min at 37°C to kill off extracellular bacteria followed by washing with 1x PBS before being lysed with 0.1% Triton-X solution (Sigma). Lysates were immediately plated for CFU counts of the 0 h time point. Fresh pH buffered DMEM + gentamicin (20 mg/l) was added to the remaining wells for later time points and incubated at 37°C with 10% CO₂. Relative intra-macrophage survival was calculated as the ratio of WT and mutant CFU/ml at 16 h divided by the ratio of WT and mutant CFU/ml at 0 h.

Cells were seeded in 12-well plates at a density of 1 × 10⁵ cells/ml and kept for 24 h incubated with a 5% CO₂ atmosphere at 37°C. After that, CML cell lines were treated with MBZ, ALB, imatinib, and dasatinib alone and in association based on IC₅₀ for 72 h. Then, cells were fixed with 70% ethanol at 4°C overnight. After the cells were centrifuged at 1500 rpm for 5 min, the supernatant was removed, and cells were incubated with 0.1% Triton-X solution (Sigma-Aldrich) for 10 min at room temperature. After new centrifugation and supernatant removal, the cells were incubated in RNAse-free DNA solution (100 μg ml⁻¹) for 20 min at 37°C. Then, propidium iodide (PI) was added directly into the RNAse solution at a final concentration of 50 μg ml⁻¹. After a 1-h incubation, cells were centrifuged again to remove the supernatant and resuspended in ×1 PBS solution. Thus, 10,000 events were analyzed by flow cytometry (BD FACSverseTM).